Taste Receptors Of The T1R Family From Domestic Cat

ABSTRACT

The present invention relates to the discovery of several genes of the domestic cat ( Felis catus ) associated with taste perception. The invention provides, inter alia, the nucleotide sequence of the feline Tas1r1, Tas1r2, and Tas1r3 receptor genes, the amino acid sequences of the polypeptides encoded thereby, and antibodies to the polypeptides. The present invention also relates to methods for screening for compounds that modify the genes&#39; function or activity, the compounds identified by such screens, and mimetics of the identified compounds. The invention further provides methods for modifying the taste preferences, ingestive responses, or general behavior of a mammal, such as a cat, by administering compounds that affect the function or activity of the gene or the polypeptide encoded thereby.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.12/406,334, filed Mar. 18, 2009, which is a divisional of U.S.application Ser. No. 10/591,360, now U.S. Pat. No. 7,527,944, which isthe U.S. National Stage of International Application No.PCT/US2004/015136, filed May 13, 2004, which claims the benefit of U.S.Provisional Application Ser. No. 60/482,992, filed Jun. 27, 2003 and ofU.S. Provisional Application Ser. No. 60/554,751, filed Mar. 19, 2004.The content of each of these applications is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates to the field of sensory mechanisms of thedomestic cat, Felis catus. The invention relates, for example, to thediscovery of several genes of Felis catus encoding taste receptors ofthe T1R family, Tas1r1, Tas1r2, and Tas1r3, the polypeptides encodedthereby (T1R1, T1R2, and T1R3), and methods and uses of the same.

BACKGROUND OF THE INVENTION

The sense of taste is important for determining food choice, forregulating food intake, and for ensuring efficient use of ingestednutrients. Taste can act as a warning system for the presence ofpotentially harmful foods, by, for example, the aversive sensations ofsourness or bitterness, and as an attractant to potentiallynutrient-rich foods, by, for example, the appealing sensations ofsweetness, saltiness, and umami.

Taste stimuli are received by taste receptor cells assembled into tastebuds that are located in the epithelium of taste papillae of the tongue(Kitagawa et al., Bioch. Bioph. Res. Comm., 283:236-242 (2001)). Thestimuli are believed to be transduced by taste receptors at the surfaceof the taste receptor cells (Id.). The taste receptors encoded by thegenes of a given species are reflective of that species' food choices.For example, the “sweet receptors” of an herbivorous species areexpected to be different from those of a carnivorous species, since thetwo consume completely different diets whose foods contain differentprimary stimuli. Since taste receptor specificity likely reflects foodchoice, it follows that receptor sequence homology among species may beas predictive or more predictive of food preferences of a given speciesas phylogenetic relatedness among species.

The behavior of the domestic cat (Felis catus), a carnivore, towardsstimuli such as sweet carbohydrates, which it generally cannot taste,and towards L-amino acids, which it generally can taste, should beexplicable by the specificity of taste receptors of other carnivores.Direct knowledge of taste receptor genes will allow insight into ananimal's sensory world and may be useful for identifying modulators ofthe taste receptors encoded thereby to influence an animal's tastepreferences.

Molecular receptors for the taste element of sweetness have beenidentified from human, mouse, and rat. Thus far, there are three knownmembers of the T1R taste receptor family: T1R1, T1R2, and T1R3(Montmayeur & Matsunami, Curr. Opin. Neurobiol., 12(4):366-371 (2002)).The T1R3 receptor gene is located within the Sac locus, the primarygenetic locus controlling preference for sweet-tasting stimuli in mice(Li et al., Mamm. Genome, 12(1):13-16 (2001); Li et al., Mamm. Genome,13(1):5-19 (2002)). The human syntenic region for mouse T1R3 gene is on1p36.33 (1162-1186 kb). The gene for T1R1 is located on human 1p36.23(6324-6349 kb), which is ˜5 Mb from T1R3, and that for T1R2 is locatedon human 1p36.13 (18483-18729 kb), which is ˜12 Mb from T1R1.

Most of the T1Rs are G-protein coupled receptors with long N-terminalextracellular domains believed to be involved in ligand binding(Montmayeur & Matsunami, Curr. Opin. Neurobiol., 12(4):366-371 (2002)).The T1R receptors have been shown to dimerize. For example,homodimerization of T1Rs has been detected (Zhao et al., Cell,115:255-266 (2003)). The taste receptors also heterodimerize. Forexample, coupling of T1R3 with T1R1 or T1R2 has been detected. In mouse,the T1R1/T1R3 heterodimer functions as a receptor for selected aminoacids. The T1R2/T1R3 heterodimer functions as a receptor for stimuliconsidered sweet by humans. Current data indicate that the T1R3component of the T1R dimer couples the taste receptor to cellular signaltransduction processes, thereby ensuring that the stimulus-binding eventis transduced to a neural signal. Thus, knowledge of the T1R receptorswill lead to better understanding of species-specific reactions to sapidstimuli.

Currently, mechanisms for identifying novel taste stimuli for thedomestic cat are limited, for example, to exhaustive and difficultfeeding studies in which a novel ingredient is paired with a controlingredient and intake of the two are compared. Considerable time,effort, and expense can be expended in the discovery of a singlestimulus. Furthermore, feline illnesses often are exacerbated by a cat'srefusal to eat. Additionally, the molecular features that defineacceptable taste stimuli for domestic cat remain largely unknown, makingrational computational design approaches for taste stimuli difficult. Asa result, knowledge of the feline taste receptor and its ligands maylead to a better understanding of cat taste perception and modulationthereof.

The present invention provides novel genes encoding the feline tastereceptors T1R1, T1R2, and T1R3, the polypeptides encoded thereby, andmethods of use of the receptors to identify compounds that canstimulate, inhibit, or modify the ingestive responses or generalbehavior of a cat. The screening methods of the invention allow therapid screening of binding partners, agonists, antagonists, andmodulators of the T1R receptors of the domestic cat. The results of thefeline T1R receptor studies reflect the unique taste profile of thedomestic cat.

SUMMARY OF THE INVENTION

Certain embodiments of the present invention relate to polynucleotidesencoding a T1R receptor, including, but not limited to polynucleotideshaving the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:99, SEQ IDNO:59, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:63, fragments of thepolynucleotide of SEQ ID NO:1, SEQ ID NO:99, SEQ ID NO:59, SEQ ID NO:60,SEQ ID NO:62, or SEQ ID NO:63 encoding a polypeptide havingsubstantially the same biological activity as a polypeptide encoded bythe nucleotide sequence of SEQ ID NO:1, SEQ ID NO:99, SEQ ID NO:59, SEQID NO:60, SEQ ID NO:62, or SEQ ID NO:63, respectively; variants of thepolynucleotide of SEQ ID NO:1 or SEQ ID NO:99 having at least 80%homology to the polynucleotide of SEQ ID NO:1 or SEQ ID NO:99; variantsof the polynucleotide of SEQ ID NO:59 or SEQ ID NO:60 having at least85% homology to the polynucleotide of SEQ ID NO:59 or SEQ ID NO:60;variants of the polynucleotide of SEQ ID NO:62 or SEQ ID NO:63 having atleast 75% homology to SEQ ID NO:62 or SEQ ID NO:63; polynucleotidevariants of SEQ ID NO:1, SEQ ID NO:99, SEQ ID NO:59, SEQ ID NO:60, SEQID NO:62, or SEQ ID NO:63 encoding a polypeptide having substantiallythe same biological activity as a polypeptide encoded by the nucleotidesequence of SEQ ID NO:1, SEQ ID NO:99, SEQ ID NO:59, SEQ ID NO:60, SEQID NO:62, or SEQ ID NO:63, respectively; variants of the polynucleotideof SEQ ID NO:1, SEQ ID NO:99, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:62,or SEQ ID NO:63 encoding a polypeptide conferring modified tasteperception to one or more taste stimuli relative to a polypeptideencoded by the polynucleotide of SEQ ID NO:1, SEQ ID NO:99, SEQ IDNO:59, SEQ ID NO:60, SEQ ID NO:62, or SEQ ID NO:63, respectively;nucleotide sequences encoding the amino acid sequence of SEQ ID NO:2,SEQ ID NO:61, or SEQ ID NO:64; nucleotide sequences substantiallycomplementary to the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:99,SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:62, or SEQ ID NO:63; andnucleotide sequences that hybridize to the complement of thepolynucleotide having SEQ ID NO:1, SEQ ID NO:99, SEQ ID NO:59, SEQ IDNO:60, SEQ ID NO:62, or SEQ ID NO:63 under high stringency conditions.The biological activity of the polypeptides encoded by thepolynucleotides of the invention may be determined, for example, by anin vitro binding assay, such as but not limited to assessing the levelof binding of the polypeptide to a T1R dimerization partner. Thepolynucleotides of the invention may be DNA or RNA and may be single- ordouble-stranded. In some embodiments of the invention, thepolynucleotide fragments have at least about 42 nucleotides. Thepolynucleotide fragments of the invention encode, for example, anextracellular domain of the polypeptide of SEQ ID NO:2, SEQ ID NO:61, orSEQ ID NO:64; a transmembrane domain of the polypeptide of SEQ ID NO:2,SEQ ID NO:61, or SEQ ID NO:64; or an intracellular domain of thepolypeptide of SEQ ID NO:2, SEQ ID NO:61, or SEQ ID NO:64. For example,the polynucleotides of the invention encoding extracellular domains offeline T1R receptors include nucleotides 1-1689, 1870-1905, 2101-2178,and 2341-2376 of SEQ ID NO:60; nucleotides 1-441 of SEQ ID NO:63; andnucleotides 1-1713, 1882-1923, 2113-2193, and 2359-2382 of SEQ ID NO:1or SEQ ID NO:99. The polynucleotides of the invention encodingtransmembrane domains of feline T1R receptors include nucleotides1690-1767, 1810-1869, 1906-1980, 2041-2100, 2179-2244, 2281-2340, and2379-2451 of SEQ ID NO:60; nucleotides 442-501 of SEQ ID NO:63; andnucleotides 1714-1782, 1828-1881, 1924-1992, 2041-2112, 2191-2262,2299-2358, and 2383-2436 of SEQ ID NO:1 or SEQ ID NO:99. Thepolynucleotides of the invention encoding intracellular domains offeline T1R receptors include nucleotides 1768-1809, 1981-2040,2245-2280, and 2452-2523 of SEQ ID NO:60; nucleotides 502-1173 of SEQ IDNO:63; nucleotides 1783-1827, 1993-2040, 2263-2298, and 2437-2566 of SEQID NO:1; and nucleotides 1783-1827, 1993-2040, 2263-2298, and 2437-2595of SEQ ID NO:99. The polynucleotides of the invention also include anycombination of the polynucleotides encoding the functional domains ofthe invention, such as combinations of the polynucleotides encoding theextracellular, transmembrane, and/or intracellular domains of the sameor different feline T1R receptors. In other embodiments of theinvention, the polynucleotide variants of the polynucleotide of SEQ IDNO:1 or SEQ ID NO:99 encoding an amino acid sequence of SEQ ID NO:2 havea nonconserved amino acid substitution, for example, at residue 59and/or residue 64.

The invention also encompasses expression vectors containing thepolynucleotides of the invention operably linked to a promoter. Anotherembodiment of the invention provides host cells containing theexpression vector. The host cells may be prokaryotic, such as bacterialcells, or eukaryotic, such as yeast or mammalian cells, including human,murine, porcine, bovine, canine, or feline cells. The invention furtherencompasses cell cultures of the host cells. The invention alsoencompasses methods of producing a feline T1R receptor by culturing thehost cells and recovering receptor therefrom.

Another embodiment of the invention includes T1R receptor polypeptides,including polypeptides encoded by the polynucleotides of the invention.The polypeptides of the invention include, for example, those having theamino acid sequence of SEQ ID NO:2, SEQ ID NO:61, or SEQ ID NO:64,fragments of at least 30 contiguous amino acids of SEQ ID NO:2, SEQ IDNO:61, or SEQ ID NO:64, and variants thereof having substantially thesame biological activity as the polypeptide of SEQ ID NO:2, SEQ IDNO:61, or SEQ ID NO:64, respectively. The biological activity of thepolypeptides of the invention may be determined, for example, by an invitro binding assay, such as but not limited to assessing the level ofbinding of the polypeptide to a T1R dimerization partner. Biologicalactivity of the polypeptides of the invention also may be determined bymeasuring ion conductance; ion flow; calcium imaging including withfura-2, green dextran activity, or aquorin activity; voltage measurementand/or voltage imaging with dyes or reporter genes such as β-luciferase,alkaline phosphatase, β-galactosidase, or β-lactamase; second messengermeasurement, for example, IP₃, cAMP, G-protein activation-based assays;or receptor phosphorylation. The variant polypeptides of the inventionmay have an amino acid sequence having at least one sequence variationof SEQ ID NO:2, SEQ ID NO:61, or SEQ ID NO:64 that confers modifiedtaste perception to one or more taste stimuli relative to a polypeptideof SEQ ID NO:2, SEQ ID NO:61, or SEQ ID NO:64, respectively. Thepolypeptides of the invention further comprise functional domains of theT1R receptors of the invention, for example, extracellular,transmembrane, and intracellular domains of the receptors, andcombinations thereof. Examples of functional domains of the T1R1polypeptide of SEQ ID NO:61 include extracellular domains correspondingto residues 1-563, 624-635, 701-726, and 781-792; transmembrane domainscorresponding to residues 564-589, 604-623, 636-660, 681-700, 727-748,761-780, and 793-817; and intracellular domains corresponding toresidues 590-603, 661-680, 749-760, and 818-841. Examples of functionaldomains of the T1R2 receptor of SEQ ID NO:64 include an extracellulardomain corresponding to residues 1-147; a transmembrane domaincorresponding to residues 148-167; and an intracellular domaincorresponding to residues 168-391. Examples of functional domains of theT1R3 polypeptide of SEQ ID NO:2 include the extracellular domains(residues 1-571, 628-641, 705-730, and 787-794 of SEQ ID NO:2), thetransmembrane domains (residues 572-594, 610-627, 642-664, 681-704,731-754, 767-780, and 795-812 of SEQ ID NO:2), and the intracellulardomains (residues 595-609, 665-680, 755-766, and 813-865 of SEQ IDNO:2). The polypeptides of the invention also include any combination ofthe functional domains of the polypeptides of the invention, such ascombinations of the extracellular, transmembrane, and/or intracellulardomains of the same or different feline T1R receptors.

The invention provides methods of identifying a feline T1R receptorvariant that confers modified taste perception by expressing a variantof the polynucleotide of SEQ ID NO:1, SEQ ID NO:99, SEQ ID NO:59, SEQ IDNO:60, SEQ ID NO:62, or SEQ ID NO:63 homologous to the polynucleotide ofSEQ ID NO:1, SEQ ID NO:99, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:62, orSEQ ID NO:63, respectively, and detecting an increase or a decrease inthe biological activity of the polypeptide encoded by the variantrelative to the biological activity of the polypeptide encoded by SEQ IDNO:1, SEQ ID NO:99, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:62, or SEQ IDNO:63, respectively.

The invention further provides kits for the detection of polynucleotidesencoding a feline T1R receptor including a polynucleotide thatspecifically hybridizes to a polynucleotide encoding a polypeptidehaving an amino acid sequence of SEQ ID NO:2, SEQ ID NO:61, or SEQ IDNO:64, and instructions relating to detection thereof.

Also provided by the invention are antibodies that immunoreactspecifically with at least one epitope of a polypeptide of theinvention. The invention also includes kits for the detection ofpolypeptides encoding a feline T1R receptor including antibodies of theinvention and instructions relating to detection.

Further provided by the invention are methods for identifying a compoundthat interacts with a feline T1R receptor by expressing a polynucleotideof the invention in the presence of a test compound, and detectingdirect or indirect interaction between a polypeptide produced by theexpression step with the compound. Also provided are methods foridentifying compounds that interact with a feline T1R receptor bycontacting a feline T1R receptor with a test compound, and detectinginteraction between the receptor and the compound. The methods fordetecting such interaction may be cell-based or cell-free assays. Forexample, a polynucleotide of the invention may be expressed in aheterologous expression system or in a cellular extract. The receptormay be bound to a solid support. In one aspect of the invention, therecognition sites of the receptor are coupled with a monitoring system,either electrical or optical. In another embodiment, the solid supportis formulated into a feline-specific electronic tongue or biosensor.

The invention also provides methods for identifying agonists andantagonists of a feline T1R receptor. For example, the methods of theinvention include identification of an agonist of a feline T1R receptorby expressing a polynucleotide of the invention in the presence of atest compound, and detecting increased transcription of saidpolynucleotide or increased biological activity of a polypeptideproduced by the expression step in the presence of the compound relativeto the rate of transcription or biological activity of the polypeptidein the absence of the compound. The biological activity detected may bean increase or decrease in the interaction between the T1R receptor andits T1R dimerization partner. For example, the T1R dimerization partnerof a T1R1 or a T1R2 receptor may be T1R3 and vice versa. (In addition,T1R receptors may form homodimers which may have unique ligand bindingproperties. The T1R dimerization partner thus also may be ahomodomerization partner. Also included are methods for identifyingagonists of a feline T1R receptor by contacting a polypeptide of theinvention with a test compound, and detecting an increase in biologicalactivity of the polypeptide in the presence of the compound relative tobiological activity of the polypeptide in the absence of the compound.The methods for identifying agonists of the cat T1R receptors may becell-based or cell-free assays. For example, a polynucleotide of theinvention may be expressed in a heterologous expression system or in acellular extract. The receptor may be bound to a solid support. In oneaspect of the invention, the recognition sites of the receptor arecoupled with a monitoring system, either electrical or optical. Inanother embodiment, the solid support is formulated into afeline-specific electronic tongue or biosensor.

Methods for identifying antagonists of the polypeptides of the inventionalso are provided. For example, the invention provides methods foridentifying antagonists of a feline T1R receptor by expressing apolynucleotide of the invention in the presence of a test compound, anddetecting decreased transcription of said polynucleotide or decreasedbiological activity of a polypeptide produced by the expression step inthe presence of the compound relative to the rate of transcription orbiological activity of the polypeptide in the absence of the compound.Another example of methods for identifying an antagonist of a feline T1Rreceptor involves contacting a polypeptide of the invention with a testcompound, and detecting a decrease in biological activity of thepolypeptide in the presence of the compound relative to biologicalactivity of the polypeptide in the absence of the compound. The methodsfor identifying the antagonists may be cell-based or cell-free assays.For example, a polynucleotide of the invention may be expressed in aheterologous expression system or in a cellular extract. The receptormay be bound to a solid support. In one aspect of the invention, therecognition sites of the receptor are coupled with a monitoring system,either electrical or optical. In another embodiment, the solid supportis formulated into a feline-specific electronic tongue or biosensor.

Also encompassed by the invention are methods for predicting the tasteperception of an organism such as a mammal, including but not limited toa cat. The methods may involve detection of a nucleotide sequence oramino acid sequence of the invention in a biological sample of theorganism. For example, an organism having a polynucleotide orpolypeptide of the invention may be attracted to one or more aminoacids. An organism having a polynucleotide or polypeptide of theinvention may show no preference for one or more carbohydrates orhigh-intensity sweeteners. An organism having a polynucleotide orpolypeptide of the invention may be attracted to one or more amino acidsand may exhibit no preference for one or more carbohydrates orhigh-intensity sweeteners.

Another embodiment of the invention includes compounds and compositionsfor modifying the taste perception of a mammal, such as a cat. Thecompounds and compositions may contain at least one of thepolynucleotides of the invention, polypeptides of the invention, orcompounds identified by the methods of the invention. Examples of thecompositions of the invention include veterinary foods and drinks andpharmaceutical compositions. The compositions of the invention mayinclude a pharmaceutically acceptable excipient. The compositions of theinvention may be breed-specific. Methods for modifying the tasteperception of a mammal (e.g., a cat) by administering to the mammal apolynucleotide of the invention, a polypeptide of the invention, and/ora compound identified according to the methods of the invention also areprovided.

The invention further contemplates transgenic animals comprising apolynucleotide of the invention.

The materials, methods, and examples provided herein are illustrativeonly and are not intended to be limiting. Other features and advantagesof the invention will be apparent from the following detaileddescription and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1I show the multiple sequence alignment of the cDNAs encodingT1R receptors of domestic cat (Tas1r1, SEQ ID NO:60; Tas1r2, SEQ IDNO:63; and Tas1r3, SEQ ID NO:99) with known nucleotide sequences ofreceptors of the T1R family from human (Tas1r1, SEQ ID NO:8; Tas1r2, SEQID NO:5; Tas1r3, SEQ ID NO:11), mouse (Tas1r1, SEQ ID NO:6; Tas1r2, SEQID NO:3; Tas1r3, SEQ ID NO:9), and rat (Tas1r1, SEQ ID NO:7; Tas1r2, SEQID NO:4; Tas1r3, SEQ ID NO:10). An asterisk (*) indicates a conservednucleotide position among the sequences. A heart (♡) indicates the stopcodon of feline T1R2.

FIGS. 2A-D show the deduced amino acid sequences of the feline T1R tastereceptors (T1R1, SEQ ID NO:61; T1R2, SEQ ID NO:64; and T1R3, SEQ IDNO:2) aligned with the amino acid sequences of members of the T1Rreceptor family from human (T1R1, SEQ ID NO:17; T1R2, SEQ ID NO:20;T1R3, SEQ ID NO:12), rat (T1R1, SEQ ID NO:16; T1R2, SEQ ID NO:19; T1R3,SEQ ID NO:14), and mouse (T1R1, SEQ ID NO:15; T1R2, SEQ ID NO:18; T1R3,SEQ ID NO:13). An asterisk (*) indicates a conserved nucleotide positionamong the sequences. A colon (:) indicates an observed conserved aminoacid substitution. A period (.) indicates an observed semi-conservedamino acid substitution. The deduced amino acid sequence for cat T1R3(SEQ ID NO:2) contains four additional amino acids at positions 11-14relative to the T1R3 receptors of mouse (SEQ ID NO:13), human (SEQ IDNO:12), and rat (SEQ ID NO:14). The deduced sequence for cat reveals athreonine in position 64, a position equivalent to amino acid 60 inmouse, and a leucine at position 59, a position equivalent to position55 in mouse. In mouse, amino acid substitutions of a threonine atposition 60 and an alanine at position 55, both positions located withinthe putative extracellular N-terminal domain of the polypeptide, arepresent in strains of mice demonstrating low preference for the sweetstimulus saccharin (Bachmanov et al., Chem. Senses, 26:925-933 (2001)).Leucine is a conservative substitution for alanine. Accordingly, theamino acid sequence differences of cat and mouse T1R3 receptor mayaccount for functional differences that lead to different tastepreferences between the two species.

FIG. 3 illustrates a phylogenetic tree showing the relatedness of thedomestic cat T1R receptor family to the T1R family of receptorsincluding human, rat, and mouse T1R1, T1R2, and T1R3. The T1R receptorsof the rat and mouse are closely related, while the T1R receptors ofhuman and cat diverge from rat and mouse. Interestingly, the sweetstimuli to which the rat and mouse respond are very similar, whereasthose that stimulate the human and those that stimulate the cat differfrom one another and from those for rat and mouse. For example, humansare unique in their ability to taste most high-intensity sweeteners,while cats find many amino acids attractive but are unable to taste mostcarbohydrate and high-intensity sweeteners. The cat T1R2 diverges fromthat of human, mouse, and rat, which is consistent with the fact thatcat does not show a preference for carbohydrate sweeteners.

FIG. 4 illustrates the predicted conformation of cat T1R3 receptor. Thecat T1R3 receptor is a seven-transmembrane domain receptor. Thestructure of the feline T1R3 receptor was generated through use of theprotein modeling program publicly available through the EuropeanBioinformatics Institute of the European Molecular Biology Laboratory.

FIG. 5A shows the predicted conformation of cat T1R1 (SEQ ID NO: 61),indicating that the receptor is a 7-transmembrane-type receptor. FIG. 5Billustrates the predicted conformation of cat T1R2 (SEQ ID NO: 64).Since feline T1R2 is a short protein (391 amino acids), a7-transmembrane domain protein is not predicted. Without seventransmembrane domains, the cat T1R2 receptor may not interactappropriately with its dimerization partner, such as T1R3, and/or theplasma membrane, which may result in the cat's indifference toward sweetcarbohydrates. The cat T1R2 may have another function.

FIGS. 6A-D show the genomic sequence of cat Tas1r1 (SEQ ID NO:59)obtained from BAC sequencing. The letter “N” denotes gaps between exonsor unknown sequences.

FIGS. 7A-E show the genomic sequence of cat Tas1r2 (SEQ ID NO:62)obtained from BAC sequencing. The letter “N” denotes gaps between exonsor unknown sequences.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The reference works, patents, patent applications, and scientificliterature that are referred to herein reflect in part the knowledge ofthose with skill in the art and are hereby incorporated by reference intheir entirety to the same extent as if each was specifically andindividually indicated to be incorporated by reference. Any conflictbetween any reference cited herein and the specific teachings of thisspecification shall be resolved in favor of the latter. Likewise, anyconflict between an art-understood definition of a word or phrase and adefinition of the word or phrase as specifically taught in thisspecification shall be resolved in favor of the latter.

Standard reference works setting forth the general principles ofrecombinant DNA technology are known to those of skill in the art(Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley &Sons, New York, 1998; Sambrook et al., MOLECULAR CLONING: A LABORATORYMANUAL, 2D ED., Cold Spring Harbor Laboratory Press, Plainview, N.Y.,1989; Kaufman et al., Eds., HANDBOOK OF MOLECULAR AND CELLULAR METHODSIN BIOLOGY AND MEDICINE, CRC Press, Boca Raton, 1995; McPherson, Ed.,DIRECTED MUTAGENESIS: A PRACTICAL APPROACH, IRL Press, Oxford, 1991).

As used herein, “T1R receptor” encompasses the taste receptors of theT1R1, T1R2, and T1R3 types, for example, the feline T1R1, T1R2, and T1R3taste receptors of the invention.

As used herein, “taste perception” refers to a response (e.g.,biochemical, behavioral) or sensitivity of a T1R receptor of theinvention to a taste stimulus. “Taste stimulus” as used herein refers toany compound that elicits, for example at the biochemical level (e.g.,activation or inhibition of a taste receptor) or behavioral level (e.g.,preference, indifference, or distaste), a taste response which would beperceived by a mammal as at least one of the five taste elements,including sweet, salty, sour, bitter, and umami. “Taste perception” or“taste stimulus,” or variants thereof, does not require, though it doesinclude, transmission of a neural signal resulting in in vivo sensationof taste by a mammal. Modification of taste perception includes analteration of (enhancement of, reduction to, or change to) a biochemicalresponse, an ingestive response, a taste preference, or general behaviorof a mammal in response to a compound.

As used herein “polynucleotide” refers to a nucleic acid molecule andincludes genomic DNA, cDNA, RNA, mRNA, mixed polymers, recombinantnucleic acids, fragments and variants thereof, and the like.Polynucleotide fragments of the invention comprise at least 10, andpreferably at least 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 75, or100 consecutive nucleotides of a reference polynucleotide. Thepolynucleotides of the invention include sense and antisense strands.The polynucleotides of the invention may be naturally occurring ornon-naturally occurring polynucleotides. A “synthesized polynucleotide”as used herein refers to polynucleotides produced by purely chemical, asopposed to enzymatic, methods. “Wholly” synthesized DNA sequences aretherefore produced entirely by chemical means, and “partially”synthesized DNAs embrace those wherein only portions of the resultingDNA were produced by chemical means. The polynucleotides of theinvention may be single- or double-stranded. The polynucleotides of theinvention may be chemically modified and may contain non-natural orderivatized nucleotide bases as will be readily appreciated by thoseskilled in the art. Such modifications include, for example, labels,methylation, substitution of one or more nucleotides with an analog,internucleotide modifications such as uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.),charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.),pendent moieties (e.g., polypeptides, etc.), intercalators (e.g.,acridine, psoralen, etc.), chelators, alkylators, and modified linkages(e.g., alpha anomeric nucleic acids, etc.). Also included are syntheticmolecules that mimic polynucleotides in their ability to bind to adesignated sequence via hydrogen bonding and other chemicalinteractions. Such molecules are known in the art and include, forexample, those in which peptide linkages substitute for phosphatelinkages in the backbone of the molecule.

“Recombinant nucleic acid” is a nucleic acid generated by combination oftwo segments of nucleotide sequence. The combination may be, forexample, by chemical means or by genetic engineering.

As used herein, “polynucleotide amplification” refers to a broad rangeof techniques for increasing the number of copies of specificpolynucleotide sequences. Typically, amplification of either or bothstrand(s) of the target nucleic acid comprises the use of one or morenucleic acid-modifying enzymes, such as a DNA polymerase, ligase, RNApolymerase, or RNA-dependent reverse transcriptase. Examples ofpolynucleotide amplification include, but are not limited to, polymerasechain reaction (PCR), nucleic acid sequence based amplification (NASB),self-sustained sequence replication (3SR), strand displacementactivation (SDA), ligase chain reaction, Qβ replicase system, and thelike. A wide variety of alternative cloning and in vitro amplificationmethodologies are well known to those skilled in the art. Examples ofthese techniques are found in, for example, Berger et al., Guide toMolecular Cloning Techniques, METHODS IN ENZYMOLOGY 152, Academic Press,Inc., San Diego, Calif. (Berger), which is incorporated herein byreference in its entirety.

As used herein, the term “oligonucleotide” or “primer” refers to aseries of linked nucleotide residues which has a sufficient number ofbases to be used in a polymerase chain reaction (PCR). This shortsequence is based on (or designed from) a genomic or cDNA sequence andis used to amplify, confirm, or reveal the presence of an identical,similar, or complementary DNA or RNA in a particular cell or tissue.Oligonucleotides comprise portions of a nucleic acid sequence having atleast about 10 nucleotides and as many as about 50 nucleotides, oftenabout 12 or 15 to about 30 nucleotides. They are chemically synthesizedand may be used as probes. “Primer pair” refers to a set of primersincluding a 5′ upstream primer that hybridizes with the 5′ end of atarget sequence to be amplified and a 3′ downstream primer thathybridizes with the complement of the 3′ end of the target sequence tobe amplified.

As used herein, the term “probe” refers to nucleic acid sequences ofvariable length, for example between at least about 10 and as many asabout 6,000 nucleotides, depending on use. Probes are used in thedetection of identical, similar, or complementary target nucleic acidsequences, which target sequences may be single- or double-stranded.Longer probes are usually obtained from a natural or recombinant source,are highly specific, and are much slower to hybridize than oligomers, orshorter probes. They may be single- or double-stranded and are carefullydesigned to have specificity in PCR, hybridization membrane-based, orELISA-like technologies. An “overgo probe” is a DNA probe comprising twoshort, overlapping DNA sequences (e.g., 10-50 nucleotides each) with acomplementary overlapping region (e.g., 5-15 nucleotides) that is usedin an overgo hybridization strategy. For example, an overgo probe may betwo 22mers with an 8 bp complementary overlap, resulting in a 36merovergo probe. As another example, an overgo probe may be two 24mers withan 8 bp complementary overlap, resulting in a 40mer overgo probe.

As used herein, the phrase “stringent hybridization conditions” or “highstringency conditions” refers to conditions under which a probe, primer,or oligonucleotide will hybridize to its target sequence, but to aminimal number of or no other sequences. Stringent conditions aresequence-dependent and will be different in different circumstances.Longer sequences will hybridize with specificity to their propercomplements at higher temperatures. Generally, stringent conditions areselected to be about 5° C. lower than the thermal melting point (T_(m))for the specific sequence at a defined ionic strength and pH. The T_(m),is the temperature (under defined ionic strength, pH and nucleic acidconcentration) at which 50% of the probes complementary to the targetsequence hybridize to the target sequence at equilibrium. Since thetarget sequences are generally present in excess, at T_(m), 50% of theprobes are hybridized to their complements at equilibrium. Stringenttemperature conditions will generally include temperatures in excess of30° C., typically in excess of 37° C., and may be in excess of 45° C.Stringent salt conditions will ordinarily be less than 1.0 M, typicallyless than 0.5 M, and may be less than 0.2 M. Typically, stringentconditions will be those in which the salt concentration is less thanabout 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (orother salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C. for short probes, primers, or oligonucleotides (e.g., 10 to 50nucleotides) and at least about 60° C. for longer probes, primers, oroligonucleotides. Stringent conditions may also be achieved with theaddition of destabilizing agents, such as formamide.

As used herein “antisense oligonucleotide” refers to a nucleic acidmolecule that is complementary to at least a portion of a targetnucleotide sequence of interest and specifically hybridizes to thetarget nucleotide sequence under physiological conditions. The term“double stranded RNA” or “dsRNA” as used herein refers to adouble-stranded RNA molecule capable of RNA interference, includingshort interfering RNA (siRNA) (see for example, Bass, Nature, 411,428-429 (2001); Elbashir et al., Nature, 411, 494-498 (2001)).

As used herein, the term “complementary” refers to Watson-Crickbasepairing between nucleotide units of a nucleic acid molecule.

The term “marker gene” or “reporter gene” refers to a gene encoding aproduct that, when expressed, confers a phenotype at the physical,morphologic, or biochemical level on a transformed cell that is easilyidentifiable, either directly or indirectly, by standard techniques andincludes, but is not limited to, genes encoding proteins that conferresistance to toxins or antibiotics such as ampicillin, neomycin, andmethotroxate; genes encoding proteins that complement auxotrophicdeficiencies; and genes encoding proteins that supply criticalcomponents not available from complex media. Examples of marker genesinclude green fluorescent protein (GFP), red fluorescent protein(DsRed), alkaline phosphatase (AP), β-lactamase, chloramphenicolacetyltransferase (CAT), adenosine deaminase (ADA), aminoglycosidephosphotransferase (neor, G418r) dihydrofolate reductase (DHFR),hygromycin-β-phosphotransferase (HPH), thymidine kinase (TK), lacZ(encoding β-galactosidase), β-lactamase, luciferase (luc), and xanthineguanine phosphoribosyltransferase (XGPRT). As with many of the standardprocedures associated with the practice of the invention, skilledartisans will be aware of additional sequences that can serve thefunction of a marker or reporter. Thus, this list is merely meant toshow examples of what can be used and is not meant to limit theinvention.

As used herein, the term “promoter” refers to a regulatory element thatregulates, controls, or drives expression of a nucleic acid molecule ofinterest and can be derived from sources such as from adenovirus, SV40,parvoviruses, vaccinia virus, cytomegalovirus, or mammalian genomic DNA.Examples of suitable promoters include, but are not limited to, CMV,MSH2, trp, lac, phage, and TRNA promoters. Suitable promoters that canbe used in yeast include, but are not limited to, such constitutivepromoters as 3-phosphoglycerate kinase and various other glycolyticenzyme gene promoters such as enolase or glyceraldehydes-3-phosphatedehydrogenase, or such inducible promoters as the alcohol dehydrogenase2 promoter or metallothionine promoter. Again, as with many of thestandard procedures associated with the practice of the invention,skilled artisans will be aware of additional promoters that can servethe function of directing the expression of a marker or reporter. Thus,the list is merely meant to show examples of what can be used and is notmeant to limit the invention.

“Operably linked” refers to juxtaposition wherein the components are ina functional relationship. For example, a promoter is operably linked orconnected to a coding sequence if it controls the transcription orexpression of the sequence.

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein. “Polypeptide” refers to a polymer of amino acidswithout referring to a specific length. Polypeptides of the inventioninclude peptide fragments, derivatives, and fusion proteins. Peptidefragments preferably have at least about 10, 15, 20, 25, 30, 35, 40, 45,50, 60, 70, 80, 90, or 100 amino acids. Some peptide fragments of theinvention are biologically active. Biological activities includeimmunogenicity, ligand binding, dimerization. and activity associatedwith the reference peptide. Immunogenic peptides and fragments of theinvention generate an epitope-specific immune response, wherein“epitope” refers to an immunogenic determinant of a peptide andpreferably contains at least three, five, eight, nine, ten, fifteen,twenty, thirty, forty, forty-five, or fifty amino acids. Someimmunogenic peptides of the invention generate an immune responsespecific to that peptide. Polypeptides of the invention includenaturally occurring and non-naturally occurring peptides. The termincludes modified polypeptides (wherein examples of such modificationsinclude glycosylation, acetylation, phosphorylation, carboxylation,ubiquitination, labeling, etc.), analogs (such as non-naturallyoccurring amino acids, substituted linkages, etc.), and functionalmimetics. A variety of methods for labeling polypeptides are well knownin the art and include radioactive isotopes such as ³²P or ³⁵S, ligandsthat bind to labeled antiligands (e.g., antibodies), fluorophores,chemiluminescent agents, enzymes, and antiligands.

As used herein, the term “amino acid” denotes a molecule containing bothan amino group and a carboxyl group. In some embodiments, the aminoacids are α-, β-, γ- or δ-amino acids, including their stereoisomers andracemates. As used herein the term “L-amino acid” denotes an α-aminoacid having the L configuration around the α-carbon, that is, acarboxylic acid of general formula CH(COOH)(NH₂)-(side chain), havingthe L-configuration. The term “D-amino acid” similarly denotes acarboxylic acid of general formula CH(COOH)(NH₂)-(side chain), havingthe D-configuration around the α-carbon. Side chains of L-amino acidsinclude naturally occurring and non-naturally occurring moieties.Non-naturally occurring (i.e., unnatural) amino acid side chains aremoieties that are used in place of naturally occurring amino acid sidechains in, for example, amino acid analogs. Amino acid substituents maybe attached, for example, through their carbonyl groups through theoxygen or carbonyl carbon thereof, or through their amino groups, orthrough functionalities residing on their side chain portions.

The amino acid sequences are presented in the amino (N) to carboxy (C)direction, from left to right. The N-terminal α-amino group and theC-terminal β-carboxy groups are not depicted in the sequence. Thenucleotide sequences are presented by single strands only, in the 5′ to3′ direction, from left to right. Nucleotides and amino acids arerepresented in the manner recommended by the IUPAC-IUB BiochemicalNomenclature Commission, or amino acids are represented by their threeletters code designations.

As used herein, the term “antibody” is meant to refer to complete,intact antibodies, and Fab, Fab′, F(ab)₂, F_(v), and other fragmentsthereof. Complete, intact antibodies include antibodies such aspolyclonal antibodies, monoclonal antibodies, chimeric antibodies, andhumanized antibodies, felinized antibodies, and immunologic bindingequivalents thereof. The antibodies of the invention may be labeled orunlabeled. Examples of labels of antibodies include, but are not limitedto, radionuclides, enzymes, substrates, cofactors, inhibitors,fluorescent agents, chemiluminescent agents, magnetic particles, and thelike. Recombinant immunoglobulins are included in the invention.

As used herein, the term “binding” means the physical or chemicalinteraction between two proteins or compounds or associated proteins orcompounds or combinations thereof. Binding includes ionic, non-ionic,Hydrogen bonds, Van der Waals, hydrophobic interactions, etc. Thephysical interaction, the binding, can be either direct or indirect,indirect being through or due to the effects of another protein orcompound. Direct binding refers to interactions that do not take placethrough or due to the effect of another protein or compound but insteadare without other substantial chemical intermediates. Binding may bedetected in many different manners. As a non-limiting example, thephysical binding interaction between two molecules can be detected usinga labeled compound. Other methods of detecting binding are well-known tothose of skill in the art.

As used herein, the term “contacting” means bringing together, eitherdirectly or indirectly, a compound into physical proximity to a moleculeof interest. Contacting may occur, for example, in any number ofbuffers, salts, solutions, or in a cell or cell extract.

As used herein, the terms “modulates” or “modifies” means an increase ordecrease in the amount, quality, or effect of a particular activity orprotein. “Modulators” refer to any inhibitory or activating moleculesidentified using in vitro and in vivo assays for, e.g., agonists,antagonists, and their homologs, including fragments, variants, andmimetics, as defined herein, that exert substantially the samebiological activity as the molecule “Inhibitors” or “antagonists” aremodulating compounds that reduce, decrease, block, prevent, delayactivation, inactivate, desensitize, or downregulate the biologicalactivity or expression of a molecule or pathway of interest. “Inducers,”“activators,” or “agonists” are modulating compounds that increase,induce, stimulate, open, activate, facilitate, enhance activation,sensitize, or upregulate a molecule or pathway of interest. In somepreferred embodiments of the invention, the level of inhibition orupregulation of the expression or biological activity of a molecule orpathway of interest refers to a decrease (inhibition or downregulation)or increase (upregulation) of greater than about 50%, 60%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. Theinhibition or upregulation may be direct, i.e., operate on the moleculeor pathway of interest itself, or indirect, i.e., operate on a moleculeor pathway that affects the molecule or pathway of interest.

A “purified” or “substantially purified” polynucleotide or polypeptideis substantially separated from other cellular components that naturallyaccompany a native (or wild-type) nucleic acid or polypeptide and/orfrom other impurities (e.g., agarose gel). A purified polypeptide orprotein will comprise about 60% to more than 99% w/w of a sample, andmay be about 90%, about 95%, or about 98% pure. As used herein, the term“isolated” refers to a molecule that has been removed from its nativeenvironment. Examples of isolated nucleic acid molecules include, butare not limited to, recombinant DNA molecules contained in a vector,recombinant DNA molecules maintained in a heterologous host cell,partially or substantially purified nucleic acid molecules, andsynthetic DNA or RNA molecules.

“About” as used herein refers to +/−10% of the reference value.

As used herein, “variant” nucleotide or amino acid sequences refer tohomologs, including, for example, isoforms, species variants, allelicvariants, and fragments of the sequence of interest. “Homologousnucleotide sequence” or “homologous amino acid sequence,” or variationsthereof, refers to sequences characterized by a homology, at thenucleotide level or amino acid level, of at least about 60%, at leastabout 70%, at least about 75%, at least about 80%, at least about 81%,at least about 82%, at least about 83%, at least about 84%, at leastabout 85%, preferably at least about 90%, at least about 95%, at leastabout 98%, or at least about 99%, and more preferably 100%, to areference sequence, or portion or fragment thereof encoding or having afunctional domain. The reference sequence may include, for example, butis not limited to the nucleic acid sequence of SEQ ID NO:1, SEQ IDNO:99, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:62, and SEQ ID NO:63, orportions thereof which encode a functional domain of the encodedpolypeptide, SEQ ID NO:2, SEQ ID NO:61, or SEQ ID NO:64, or thepolypeptide having amino acid sequence SEQ ID NO:2, SEQ ID NO:61, or SEQID NO:64, or fragments thereof having functional domains of thefull-length polypeptide. Functional domains of the T1R receptors of theinvention include extracellular domains, transmembrane domains, andintracellular domains. Examples of functional domains of the T1R1polypeptide of SEQ ID NO:61 include extracellular domains correspondingto residues 1-563, 624-635, 701-726, and 781-792; transmembrane domainscorresponding to residues 564-589, 604-623, 636-660, 681-700, 727-748,761-780, and 793-817; and intracellular domains corresponding toresidues 590-603, 661-680, 749-760, and 818-841. Examples of functionaldomains of the T1R2 receptor of SEQ ID NO:64 include an extracellulardomain corresponding to residues 1-147; a transmembrane domaincorresponding to residues 148-167; and an intracellular domaincorresponding to residues 168-391. Examples of functional domains of theT1R3 polypeptide of SEQ ID NO:2 include the extracellular domains(residues 1-571, 628-641, 705-730, and 787-794 of SEQ ID NO:2), thetransmembrane domains (residues 572-594, 610-627, 642-664, 681-704,731-754, 767-780, and 795-812 of SEQ ID NO:2), and the intracellulardomains (residues 595-609, 665-680, 755-766, and 813-865 of SEQ IDNO:2). Isoforms can be expressed in different tissues of the sameorganism as a result of, for example, alternative splicing of RNA.Alternatively, isoforms can be encoded by different genes. Homologousnucleotide sequences include nucleotide sequences encoding for a speciesvariant of a protein. Homologous nucleotide sequences also include, butare not limited to, naturally occurring allelic variations and mutationsof the nucleotide sequences set forth herein. Study of mutations andpolymorphisms of the T1R receptor polynucleotide sequences may explainbreed-specific and/or individual taste preferences of a mammal such as acat. Additionally, sequence variants of the T1R receptors may beassociated with specific disease states, such that knowledge of thegenes allows diagnosis and treatment of T1R-associated disorders (e.g.,obesity, diabetes). Homologous amino acid sequences include those aminoacid sequences which encode conservative amino acid substitutions inpolypeptides having an amino acid sequence of SEQ ID NO:2, SEQ ID NO:61,or SEQ ID NO:64, as well as in polypeptides identified according to themethods of the invention. Percent homology may be determined by, forexample, the Gap program (Wisconsin Sequence Analysis Package, Version 8for Unix, Genetics Computer Group, University Research Park, MadisonWis.), using the default settings, which uses the algorithm of Smith andWaterman (Smith and Waterman, Adv. Appl. Math., 2: 482-489, 1981).Nucleic acid fragments of the invention preferably have at least about5, at least about 10, at least about 15, at least about 20, at leastabout 25, at least about 50, or at least about 100 nucleotides of thereference nucleotide sequence. The nucleic acid fragments of theinvention may encode a polypeptide having at least one biologicalproperty, or function, that is substantially similar to a biologicalproperty of the polypeptide encoded by the full-length nucleic acidsequence.

As is well known in the art, because of the degeneracy of the geneticcode, there are numerous DNA and RNA molecules that can code for thesame polypeptide as that encoded by a nucleotide sequence of interest.The present invention, therefore, contemplates those other DNA and RNAmolecules which, on expression, encode a polypeptide encoded by thenucleic acid molecule of interest. For example, nucleotide “insertions”,“substitutions” or “deletions” are changes to or within a nucleotidesequence. Such polynucleotide variants are within the scope of theinvention. DNA and RNA molecules other than those specifically disclosedherein characterized simply by a change in a codon for a particularamino acid, are within the scope of this invention.

Amino acid “insertions”, “substitutions” or “deletions” are changes toor within an amino acid sequence. The variation allowed in a particularamino acid sequence may be experimentally determined by producing thepeptide synthetically or by systematically making insertions, deletions,or substitutions of nucleotides in the nucleic acid sequence usingrecombinant DNA techniques. Alterations of the naturally occurring aminoacid sequence can be accomplished by any of a number of knowntechniques. For example, mutations can be introduced into thepolynucleotide encoding a polypeptide at particular locations byprocedures well known to the skilled artisan, such asoligonucleotide-directed mutagenesis.

A polypeptide variant of the present invention may exhibit substantiallythe biological activity of a naturally occurring reference polypeptide.“Biological activity” as used herein refers to the level of a particularfunction (for example, enzymatic activity) of a molecule or pathway ofinterest in a biological system. “Wild-type biological activity” refersto the normal level of function of a molecule or pathway of interest.“Reduced biological activity” refers to a decreased level of function ofa molecule or pathway of interest relative to a reference level ofbiological activity of that molecule or pathway. For example, reducedbiological activity may refer to a decreased level of biologicalactivity relative to the wild-type biological activity of a molecule orpathway of interest. “Increased biological activity” refers to anincreased level of function of a molecule or pathway of interestrelative to a reference level of biological activity of that molecule orpathway. For example, increased biological activity may refer to anincreased level of biological activity relative to the wild-typebiological activity of a molecule or pathway of interest. Reference toexhibiting “substantially the biological activity of a naturallyoccurring polypeptide” indicates that variants within the scope of theinvention can comprise conservatively substituted sequences, meaningthat one or more amino acid residues of a polypeptide are replaced bydifferent residues that do not alter the secondary and/or tertiarystructure of the polypeptide. Such substitutions may include thereplacement of an amino acid by a residue having similar physicochemicalproperties, such as substituting one aliphatic residue (Ile, Val, Leu orAla) for another, or substitution between basic residues Lys and Arg,acidic residues Glu and Asp, amide residues Gln and Asn, hydroxylresidues Ser and Tyr, or aromatic residues Phe and Tyr. Furtherinformation regarding making phenotypically silent amino acid exchangesare known in the art (Bowie et al., Science, 247: 1306-1310, 1990).Other polypeptide homologs which might retain substantially thebiological activities of the reference polypeptide are those where aminoacid substitutions have been made in areas outside functional regions ofthe protein. The biological activity may be assessed by, for example,measuring binding of a T1R receptor of the invention to a dimerizationpartner. Biological activity of the polypeptides of the invention alsomay be determined by measuring ion conductance; ion flow; calciumimaging including with fura-2, green dextran activity, or aquorinactivity; voltage measurement and/or voltage imaging with dyes orreporter genes such as β-luciferase, alkaline phosphatase,β-galactosidase, or β-lactamase; second messenger measurement, forexample, IP₃, cAMP, G-protein activation-based assays; or receptorphosphorylation.

A nucleotide and/or amino acid sequence of a nucleic acid molecule orpolypeptide employed in the invention or of a compound identified by thescreening method of the invention may be used to search a nucleotide andamino acid sequence databank for regions of similarity using GappedBLAST (Altschul et al., Nuc. Acids Res., 25: 3389, 1997). Briefly, theBLAST algorithm, which stands for Basic Local Alignment Search Tool issuitable for determining sequence similarity (Altschul et al., J Mol.Biol., 215: 403-410, 1990). Software for performing BLAST analyses ispublicly available through the National Center for BiotechnologyInformation. This algorithm involves first identifying high scoringsequence pair (HSPs) by identifying short words of length W in the querysequence that either match or satisfy some positive-valued thresholdscore T when aligned with a word of the same length in a databasesequence. T is referred to as the neighborhood word score threshold(Altschul et al., J Mol. Biol., 215: 403-410, 1990). These initialneighborhood word hits act as seeds for initiating searches to find HSPscontaining them. The word hits are extended in both directions alongeach sequence for as far as the cumulative alignment score can beincreased. Extension for the word hits in each direction are haltedwhen: 1) the cumulative alignment score falls off by the quantity X fromits maximum achieved value; 2) the cumulative score goes to zero orbelow, due to the accumulation of one or more negative-scoring residuealignments; or 3) the end of either sequence is reached. The BLASTalgorithm parameters W, T, and X determine the sensitivity and speed ofthe alignment. The BLAST program uses as defaults a word length (W) of11, the BLOSUM62 scoring matrix (Henikoff et al., Proc. Natl. Acad. Sci.USA, 89: 10915-10919, 1992) alignments (B) of 50, expectation (E) of 10,M=5, N=4, and a comparison of both strands. The BLAST algorithm (Karlinet al., Proc. Natl. Acad. Sci. USA, 90: 5873-5787, 1993) and GappedBLAST perform a statistical analysis of the similarity between twosequences. One measure of similarity provided by the BLAST algorithm isthe smallest sum probability (P(N)), which provides an indication of theprobability by which a match between two nucleotide or amino acidsequences would occur by chance. For example, a nucleic acid isconsidered similar to a gene or cDNA if the smallest sum probability incomparison of the test nucleic acid to the reference nucleic acid isless than about 1, preferably less than about 0.1, more preferably lessthan about 0.01, and most preferably less than about 0.001.

The term “mimetic” as used herein refers to a compound that issterically similar to a reference compound. Mimetics are structural andfunctional equivalents to the reference compounds.

The terms “patient” and “subject” are used interchangeably herein andinclude, but are not limited to, avians, felines, canines, bovines,ovines, porcines, equines, rodents, simians, and humans. “Host cell”includes, for example, a prokaryotic cell, such as a bacterial cell, oreukaryotic cell, such as a mammalian cell (e.g., human, rodent, canine,feline), a yeast cell, or a plant cell. “Rodents” include, for example,rats and mice.

The term “treatment” as used herein refers to any indicia of success ofprevention, treatment, or amelioration of a disease or condition.Treatment includes any objective or subjective parameter, such as, butnot limited to, abatement, remission, normalization of receptoractivity, reduction in the number or severity of symptoms or sideeffects, or slowing of the rate of degeneration or decline of thepatient. Treatment also includes a prevention of the onset of symptomsin a patient that may be at increased risk for or is suspected of havinga disease or condition but does not yet experience or exhibit symptomsthereof.

As used herein, the term “compound” means any identifiable chemical ormolecule, including, but not limited to a small molecule, peptide,protein, sugar, nucleotide, or nucleic acid. Such compound can benatural or synthetic.

Topologically, sensory GPCRs have various domains including one or moreof an “N-terminal domain,” an “extracellular domain,” “transmembranedomain,” cytoplasmic and extracellular loops, “intracellular domain,”and a “C-terminal domain” (see, e.g., Hoon et al., Cell 96:541-551(1999); Buck & Axel, Cell 65:175-187 (1991)). These domains can bestructurally identified using methods known to those of skill in theart, such as sequence analysis programs that identify hydrophobic andhydrophilic domains (see, e.g., Stryer, Biochemistry (3rd ed. 1988).Such domains are useful for making chimeric proteins and for in vitroassays of the invention, e.g., ligand binding assays. In thepolypeptides of the invention, the N-terminal domain is believed to beextracellular, while the C-terminal domain is believed to be cytoplasmicor intracellular.

Polynucleotides

The invention provides purified and isolated polynucleotides (e.g.,cDNA, genomic DNA, synthetic DNA, RNA, or combinations thereof, whethersingle- or double-stranded) that comprise a nucleotide sequence encodingthe amino acid sequence of the polypeptides of the invention. Suchpolynucleotides are useful for recombinantly expressing the receptor andalso for detecting expression of the receptor in cells (e.g., usingNorthern hybridization and in situ hybridization assays). Suchpolynucleotides also are useful in the design of antisense and othermolecules for the suppression of the expression of a T1R receptor in acultured cell, a tissue, or an animal; for therapeutic purposes; or toprovide a model for diseases or conditions characterized by aberrant T1Rexpression. Specifically excluded from the definition of polynucleotidesof the invention are entire isolated, non-recombinant native chromosomesof host cells. Polynucleotides of the invention include the nucleotidesequence of SEQ ID NO:1, SEQ ID NO:99, SEQ ID NO:59, SEQ ID NO:60, SEQID NO:62, and SEQ ID NO:63. It will be appreciated that numerous otherpolynucleotide sequences exist that also encode the T1R receptors of theinvention due to the well-known degeneracy of the universal geneticcode. Such polynucleotides are included within the scope of theinvention.

The invention also provides a purified and isolated polynucleotidecomprising a nucleotide sequence that encodes a mammalian (e.g., feline)polypeptide, wherein the polynucleotide hybridizes to a polynucleotidehaving a sequence of SEQ ID NO:1, SEQ ID NO:99, SEQ ID NO:59, SEQ IDNO:60, SEQ ID NO:62, or SEQ ID NO:63 or the non-coding strandcomplementary thereto, under high stringency conditions.

Genomic DNA of the invention comprises the protein-coding region for apolypeptide of the invention and is also intended to include allelicvariants thereof. It is widely understood that, for many genes, genomicDNA is transcribed into RNA transcripts that undergo one or moresplicing events wherein introns (i.e., non-coding regions) of thetranscripts are removed, or “spliced out.” RNA transcripts that can bespliced by alternative mechanisms, and therefore be subject to removalof different RNA sequences but still encode a T1R polypeptide, arereferred to in the art as splice variants which are embraced by theinvention. Splice variants comprehended by the invention therefore areencoded by the same original genomic DNA sequences but arise fromdistinct mRNA transcripts. Allelic variants are modified forms of awild-type gene sequence, the modification resulting from recombinationduring chromosomal segregation or exposure to conditions which give riseto genetic mutation. Allelic variants, like wild type genes, arenaturally occurring sequences (as opposed to non-naturally occurringvariants that arise from in vitro manipulation).

The invention also comprehends cDNA that is obtained through reversetranscription of an RNA polynucleotide encoding a T1R receptor(conventionally followed by second strand synthesis of a complementarystrand to provide a double-stranded DNA).

One embodiment of the DNA of the invention comprises a double-strandedmolecule along with the complementary molecule (the “non-coding strand”or “complement”) having a sequence unambiguously deducible from thecoding strand according to Watson-Crick base-pairing rules for DNA.

The present invention includes fragments of nucleotide sequencesencoding a T1R receptor comprising at least 10, and preferably at least12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 75, or 100 consecutivenucleotides of a polynucleotide encoding a T1R receptor. Fragmentpolynucleotides of the invention may comprise sequences unique to theT1R-encoding polynucleotide sequence or sequences shared only by thefeline T1R-encoding polynucleotide sequences of the invention, andtherefore hybridize under highly stringent or moderately stringentconditions only (i.e., “specifically”) to polynucleotides encoding afeline T1R receptor (or fragments thereof). Polynucleotide fragments ofgenomic sequences of the invention comprise not only sequences unique tothe coding region, but also include fragments of the full-lengthsequence derived from introns, regulatory regions, and/or othernon-translated sequences. Sequences unique to polynucleotides of theinvention are recognizable through sequence comparison to other knownpolynucleotides, and can be identified through use of alignment programsroutinely utilized in the art, e.g., those made available in publicsequence databases. Such sequences also are recognizable from Southernhybridization analyses to determine the number of fragments of genomicDNA to which a polynucleotide will hybridize. Polynucleotides of theinvention can be labeled in a manner that permits their detection,including radioactive, fluorescent, and enzymatic labeling.Polynucleotide fragments of the invention also include nucleotidesequences encoding functional domains of the polypeptides of theinvention. For example, the polynucleotides of the invention encodingextracellular domains of feline T1R receptors include nucleotides1-1689, 1870-1905, 2101-2178, and 2341-2376 of SEQ ID NO:60; nucleotides1-441 of SEQ ID NO:63; and nucleotides 1-1713, 1882-1923, 2113-2193, and2359-2382 of SEQ ID NO:1 or SEQ ID NO:99. The polynucleotides of theinvention encoding transmembrane domains of feline T1R receptors includenucleotides 1690-1767, 1810-1869, 1906-1980, 2041-2100, 2179-2244,2281-2340, and 2379-2451 of SEQ ID NO:60; nucleotides 442-501 of SEQ IDNO:63; and nucleotides 1714-1782, 1828-1881, 1924-1992, 2041-2112,2191-2262, 2299-2358, and 2383-2436 of SEQ ID NO:1 or 99. Thepolynucleotides of the invention encoding intracellular domains offeline T1R receptors include nucleotides 1768-1809, 1981-2040,2245-2280, and 2452-2523 of SEQ ID NO:60; nucleotides 502-1173 of SEQ IDNO:63; nucleotides 1783-1827, 1993-2040, 2263-2298, and 2437-2566 of SEQID NO:1; and nucleotides 1783-1827, 1993-2040, 2263-2298, and 2437-2595of SEQ ID NO:99. The polynucleotides of the invention also include anycombination of the polynucleotides encoding the functional domains ofthe invention, such as combinations of the polynucleotides encoding theextracellular, transmembrane, and/or intracellular domains of the sameor different feline T1R receptors.

Fragment polynucleotides are particularly useful as probes for detectionof full-length or fragments of T1R polynucleotides. One or morepolynucleotides can be included in kits that are used to detect thepresence of a polynucleotide encoding a T1R receptor, or used to detectvariations in a polynucleotide sequence encoding a T1R receptor, forexample a feline T1R receptor.

The invention also embraces DNAs encoding T1R polypeptides thathybridize under high stringency conditions to the non-coding strand, orcomplement, of the polynucleotides.

Exemplary highly stringent hybridization conditions are as follows:hybridization at 42° C. in a hybridization solution comprising 50%formamide, 1% SDS, 1 M NaCl, 10% Dextran sulfate, and washing twice for30 minutes at 60° C. in a wash solution comprising 0.1×SSC and 1% SDS.It is understood in the art that conditions of equivalent stringency canbe achieved through variation of temperature and buffer, or saltconcentration as described, for example, in Ausubel et al. (Eds.),PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons (1994), pp. 6.0.3 to6.4.10. Modifications in hybridization conditions can be empiricallydetermined or precisely calculated based on the length and thepercentage of guanosine/cytosine (GC) base pairing of the probe. Thehybridization conditions can be calculated as described, for example, inSambrook et al., (Eds.), MOLECULAR CLONING: A LABORATORY MANUAL, ColdSpring Harbor Laboratory Press: Cold Spring Harbor, N.Y. (1989), pp.9.47 to 9.51.

With the knowledge of the nucleotide sequence information disclosed inthe present invention, one skilled in the art can identify and obtainnucleotide sequences which encode T1R receptors from different sources(i.e., different tissues or different organisms) through a variety ofmeans well known to the skilled artisan and as disclosed by, forexample, Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, SecondEdition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989).

For example, DNA that encodes a T1R receptor may be obtained byscreening mRNA, cDNA, or genomic DNA with oligonucleotide probesgenerated from the T1R gene sequence information provided herein. Probesmay be labeled with a detectable group, such as a fluorescent group, aradioactive atom or a chemiluminescent group in accordance withprocedures known to the skilled artisan and used in conventionalhybridization assays, as described by, for example, Sambrook et al.

A nucleic acid molecule comprising a T1R nucleotide sequence canalternatively be synthesized by use of the polymerase chain reaction(PCR) procedure, with the PCR oligonucleotide primers produced from thenucleotide sequences provided herein. The PCR reaction provides a methodfor selectively increasing the concentration of a particular nucleicacid sequence even when that sequence has not been previously purifiedand is present only in a single copy in a particular sample. The methodcan be used to amplify either single- or double-stranded DNA. Theessence of the method involves the use of two oligonucleotide probes toserve as primers for the template-dependent, polymerase mediatedreplication of a desired nucleic acid molecule.

A wide variety of alternative cloning and in vitro amplificationmethodologies are well known to those skilled in the art. Examples ofthese techniques are found in, for example, Berger et al., Guide toMolecular Cloning Techniques, METHODS IN ENZYMOLOGY 152, Academic Press,Inc., San Diego, Calif. (Berger), which is incorporated herein byreference in its entirety.

The polynucleotides of the invention may be used in hybridizationtechniques known to those skilled in the art, including but not limitedto, Northern and Southern blotting and overgo hybridization (see infra).For example, polynucleotide probes of the invention may be used intissue distribution studies and diagnostic assays. The T1R receptors ofthe invention are likely to be present and active in tissues other thanthose involved in taste perception. It is therefore likely that thefeline T1R receptors serve multiple functions in vivo, such as, forexample, regulation of amino acid metabolism in addition to tasteperception.

Automated sequencing methods can be used to obtain or verify the T1Rreceptor-encoding nucleotide sequence. The nucleotide sequences of thepresent invention are believed to be accurate. However, as is known inthe art, nucleotide sequences obtained by automated methods may containsome errors. Nucleotide sequences determined by automation are typicallyat least about 90%, more typically at least about 95% to at least about99.9% identical to the actual nucleotide sequence of a given nucleicacid molecule. The actual sequence may be more precisely determinedusing manual sequencing methods, which are well known in the art. Anerror in a sequence which results in an insertion or deletion of one ormore nucleotides may result in a frame shift in translation such thatthe predicted amino acid sequence will differ from that which would bepredicted from the actual nucleotide sequence of the nucleic acidmolecule, starting at the point of the mutation.

The nucleic acid molecules of the present invention, and fragmentsderived therefrom, are useful for screening for restriction fragmentlength polymorphisms (RFLP) associated with certain disorders, forgenetic mapping, and for methods for predicting the taste perception ofan organism such as a mammal involving detection of a nucleotidesequence of the invention in a biological sample of the organism. Forexample, a mammal having a polynucleotide of the invention may beattracted to the taste of one or more amino acids and/or be insensitiveor indifferent to one or more carbohydrate or high-intensity sweeteners.

The polynucleotide sequence information provided by the invention makespossible large-scale expression of the encoded polypeptide by techniqueswell known and routinely practiced in the art.

Vectors

Another aspect of the present invention is directed to vectors, orrecombinant expression vectors, comprising any of the nucleic acidmolecules described above. Vectors are used herein either to amplify DNAor RNA encoding a T1R receptor and/or to express DNA which encodes a T1Rreceptor. Examples of vectors include, but are not limited to, plasmids,phages, cosmids, episomes, viral particles or viruses, and integratableDNA fragments (i.e., fragments integratable into the host genome byhomologous recombination). Examples of viral particles include, but arenot limited to, adenoviruses, baculoviruses, parvoviruses,herpesviruses, poxviruses, adeno-associated viruses, Semliki Forestviruses, vaccinia viruses, and retroviruses. Examples of expressionvectors include, but are not limited to, pcDNA3 (Invitrogen) and pSVL(Pharmacia Biotech). Other expression vectors include, but are notlimited to, pSPORT™ vectors, pGEM™ vectors (Promega), pPROEXvectors™(LTI, Bethesda, Md.), Bluescript™ vectors (Stratagene), pQE™ vectors(Qiagen), pSE420™ (Invitrogen), and pYES2™ (Invitrogen).

Expression constructs may comprise T1R-encoding polynucleotides operablylinked to an endogenous or exogenous expression control DNA sequence anda transcription terminator. Expression control DNA sequences includepromoters, enhancers, operators, and regulatory element binding sitesgenerally, and are typically selected based on the expression systems inwhich the expression construct is to be utilized. Promoter and enhancersequences are generally selected for the ability to increase geneexpression, while operator sequences are generally selected for theability to regulate gene expression. Expression constructs of theinvention may also include sequences encoding one or more selectablemarkers that permit identification of host cells bearing the construct.Expression constructs may also include sequences that facilitate, orpromote, homologous recombination in a host cell. Constructs of theinvention also may include sequences necessary for replication in a hostcell.

Expression constructs may be utilized for production of an encodedprotein, but may also be utilized simply to amplify a T1R-encodingpolynucleotide sequence. In some embodiments, the vector is anexpression vector wherein a polynucleotide of the invention is operablylinked to a polynucleotide comprising an expression control sequence.Autonomously replicating recombinant expression constructs such asplasmid and viral DNA vectors incorporating polynucleotides of theinvention are also provided. Some expression vectors are replicable DNAconstructs in which a DNA sequence encoding a T1R receptor is operablylinked or connected to suitable control sequence(s) capable of effectingthe expression of the receptor in a suitable host. Amplification vectorsdo not require expression control domains, but rather need only theability to replicate in a host, such as conferred by an origin ofreplication, and a selection gene to facilitate recognition oftransformants. The need for control sequences in the expression vectorwill vary depending upon the host selected and the transformation methodchosen. Control sequences include a transcriptional promoter, anoptional operator sequence to control transcription, a sequence encodingsuitable mRNA ribosomal binding, and sequences which control thetermination of transcription and translation.

Vectors of the invention may contain a promoter that is recognized bythe host organism. The promoter sequences of the present invention maybe prokaryotic, eukaryotic, or viral. Examples of suitable prokaryoticsequences include the P_(R) and P_(L) promoters of bacteriophage lambda(THE BACTERIOPHAGE LAMBDA, Hershey, A. D., Ed., Cold Spring HarborPress, Cold Spring Harbor, N.Y. (1973), which is incorporated herein byreference in its entirety; LAMBDA II, Hendrix, R. W., Ed., Cold SpringHarbor Press, Cold Spring Harbor, N.Y. (1980), which is incorporatedherein by reference in its entirety), the trp, recA, heat shock, andlacZ promoters of E. coli, and the SV40 early promoter (Benoist et al.Nature, 1981, 290, 304-310), which is incorporated herein by referencein its entirety. Additional promoters include, but are not limited to,mouse mammary tumor virus, long terminal repeat of humanimmunodeficiency virus, maloney virus, cytomegalovirus immediate earlypromoter, Epstein Barr virus, Rous sarcoma virus, human actin, humanmyosin, human hemoglobin, human muscle creatine, and humanmetallothionein.

Additional regulatory sequences can also be included in vectors of theinvention. Examples of suitable regulatory sequences are represented bythe Shine-Dalgarno of the replicase gene of the phage MS-2 and of thegene cII of bacteriophage lambda. The Shine-Dalgarno sequence may bedirectly followed by DNA encoding a T1R receptor, resulting in theexpression of the mature protein.

Moreover, suitable expression vectors can include an appropriate markerthat allows the screening of transformed host cells. The transformationof the selected host is carried out using any one of the varioustechniques well known to the expert in the art and described in Sambrooket al., supra.

An origin of replication or autonomously replicating sequence (ARS) canalso be provided either by construction of the vector to include anexogenous origin or may be provided by the host cell chromosomalreplication mechanism. If the vector is integrated into the host cellchromosome, the latter may be sufficient. Alternatively, rather thanusing vectors which contain viral origins of replication, one skilled inthe art can transform mammalian cells by the method of co-transformationwith a selectable marker and T1R DNA. An example of a suitable marker isdihydrofolate reductase (DHFR) or thymidine kinase (see U.S. Pat. No.4,399,216).

Additional regulatory sequences that may be included in thepolynucleotides of the invention include secretion signals which allowthe encoded polypeptide to cross and/or lodge in cell membranes, or besecreted from the cell.

Nucleotide sequences encoding a T1R receptor may be recombined withvector DNA in accordance with conventional techniques, includingblunt-ended or staggered-ended termini for ligation, restriction enzymedigestion to provide appropriate termini, filling in of cohesive ends asappropriate, alkaline phosphatase treatment to avoid undesirablejoining, and ligation with appropriate ligases. Techniques for suchmanipulation are disclosed by Sambrook et al., supra and are well knownin the art. Methods for construction of mammalian expression vectors aredisclosed in, for example, Okayama et al., Mol. Cell. Biol., 1983, 3,280, Cosman et al., Mol. Immunol., 1986, 23, 935, Cosman et al., Nature,1984, 312, 768, EP-A-0367566, and WO 91/18982, each of which isincorporated herein by reference in its entirety.

Vectors of the invention are useful for expressing T1Rs in various cellsystems. Overexpression of a T1R may, for example, be useful inscreening for antagonists of the receptor as described herein.Stimulation of transcription of T1R polynucleotides may be used toanalyze the effect of T1R expression on the expression of other tastereceptors. Vectors may also be used to produce antisense polynucleotidesthat inhibit endogenous T1R expression to analyze the effect of a lossof the T1R gene.

Host Cells

According to another aspect of the invention, host cells are provided,including prokaryotic and eukaryotic cells, comprising a polynucleotideof the invention (or vector of the invention) in a manner that permitsexpression of the encoded T1R polypeptide. Polynucleotides of theinvention may be introduced into the host cell as part of a circularplasmid, or as linear DNA comprising an isolated protein-coding regionor a viral vector. Methods for introducing DNA into the host cell thatare well known and routinely practiced in the art includetransformation, transfection, electroporation, nuclear injection, orfusion with carriers such as liposomes, micelles, ghost cells, andprotoplasts. Expression systems of the invention include bacterial,yeast, fungal, plant, insect, invertebrate, vertebrate, and mammaliancell systems.

The invention provides host cells that are transformed or transfected(stably or transiently) with polynucleotides of the invention or vectorsof the invention. As stated above, such host cells are useful foramplifying the polynucleotides and also for expressing a T1R polypeptideor fragment thereof encoded by the polynucleotide.

In still another related embodiment, the invention provides a method forproducing a T1R polypeptide (or fragment thereof) comprising the stepsof growing a host cell of the invention in a nutrient medium andisolating the polypeptide or variant thereof from the cell or themedium. Because the T1R receptor is a membrane-spanning polypeptide, itwill be appreciated that, for some applications, such as certainactivity assays, the preferable isolation may involve isolation of cellmembranes containing the polypeptide embedded therein, whereas for otherapplications a more complete isolation may be preferable.

According to some aspects of the present invention, transformed hostcells having an expression vector comprising any of the nucleic acidmolecules described above are provided. Expression of the nucleotidesequence occurs when the expression vector is introduced into anappropriate host cell. Suitable host cells for expression of thepolypeptides of the invention include, but are not limited to,prokaryotes, yeast, and eukaryotes. If a prokaryotic expression vectoris employed, then the appropriate host cell would be any prokaryoticcell capable of expressing the cloned sequences. Suitable prokaryoticcells include, but are not limited to, bacteria of the generaEscherichia, Bacillus, Salmonella, Pseudomonas, Streptomyces, andStaphylococcus.

If a eukaryotic expression vector is employed, then the appropriate hostcell would be any eukaryotic cell capable of expressing the clonedsequence. Eukaryotic cells may be cells of higher eukaryotes. Suitableeukaryotic cells include, but are not limited to, non-human mammaliantissue culture cells and human tissue culture cells. Host cells include,but are not limited to, insect cells, HeLa cells, Chinese hamster ovarycells (CHO cells), African green monkey kidney cells (COS cells), humanHEK-293 cells, and murine 3T3 fibroblasts. Propagation of such cells incell culture has become a routine procedure (see, TISSUE CULTURE,Academic Press, Kruse and Patterson, eds. (1973), which is incorporatedherein by reference in its entirety).

In addition, a yeast host may be employed as a host cell. Yeast cellsinclude, but are not limited to, the genera Saccharomyces, Pichia, andKluveromyces. Yeast hosts may be S. cerevisiae and P. pastoris. Yeastvectors may contain an origin of replication sequence from a 2T yeastplasmid, an autonomously replication sequence (ARS), a promoter region,sequences for polyadenylation, sequences for transcription termination,and a selectable marker gene. Shuttle vectors for replication in bothyeast and E. coli are also included herein.

Alternatively, insect cells may be used as host cells. In someembodiments, the polypeptides of the invention are expressed using abaculovirus expression system (see, Luckow et al., Bio/Technology, 1988,6, 47; BACULOVIRUS EXPRESSION VECTORS: A LABORATORY MANUAL, O'Reilly etal. (Eds.), W.H. Freeman and Company, New York, 1992; and U.S. Pat. No.4,879,236, each of which is incorporated herein by reference in itsentirety). In addition, the MAXBAC™ complete baculovirus expressionsystem (Invitrogen) can, for example, be used for production in insectcells.

Host cells of the invention are a valuable source of immunogen fordevelopment of antibodies specifically immunoreactive with the T1Rreceptor. Host cells of the invention also are useful in methods for thelarge-scale production of T1R polypeptides wherein the cells are grownin a suitable culture medium and the desired polypeptide products areisolated from the cells, or from the medium in which the cells aregrown, by purification methods known in the art, e.g., conventionalchromatographic methods including immunoaffinity chromatography,receptor affinity chromatography, hydrophobic interactionchromatography, lectin affinity chromatography, size exclusionfiltration, cation or anion exchange chromatography, high pressureliquid chromatography (HPLC), reverse phase HPLC, and the like. Stillother methods of purification include those methods wherein the desiredprotein is expressed and purified as a fusion protein having a specifictag, label, or chelating moiety that is recognized by a specific bindingpartner or agent. The purified protein can be cleaved to yield thedesired protein, or can be left as an intact fusion protein. Cleavage ofthe fusion component may produce a form of the desired protein havingadditional amino acid residues as a result of the cleavage process.

Knowledge of the feline T1R receptor-encoding nucleotide sequence allowsfor modification of cells to permit, or increase, expression ofendogenous receptor. Cells can be modified (e.g., by homologousrecombination) to provide increased expression by replacing, in whole orin part, the naturally occurring T1R promoter with all or part of aheterologous promoter so that the cells express the receptor at higheror lower levels. The heterologous promoter is inserted in such a mannerthat it is operably linked to endogenous T1R coding sequence. (See, forexample, PCT International Publication No. WO 94/12650, PCTInternational Publication No. WO 92/20808, and PCT InternationalPublication No. WO 91/09955.) It is also contemplated that, in additionto heterologous promoter DNA, amplifiable marker DNA (e.g., ada, dhfr,and the multifunctional CAD gene which encodes carbamoyl phosphatesynthase, aspartate transcarbamylase, and dihydroorotase) and/or intronDNA may be inserted along with the heterologous promoter DNA. If linkedto the T1R coding sequence, amplification of the marker DNA by standardselection methods results in co-amplification of the T1R codingsequences in the cells.

Knock-Out and Transplacement Animals

The DNA sequence information provided by the present invention alsomakes possible the development (e.g., by homologous recombinationstrategies; see Capecchi, Science 244:1288-1292 (1989), which isincorporated herein by reference) of transgenic or gene-targetedanimals, including, for example, animals that fail to express functionalT1R (“knock-out”) or that express a variant thereof (“transplacement’).Such animals (especially small laboratory animals such as rats, rabbits,mice, and cats) are useful as models for studying the in vivo activitiesof T1R receptors and modulators of T1R receptors.

Antisense and siRNA

Also encompassed by the invention are antisense and short interferingpolynucleotides that recognize and hybridize to polynucleotides encodingthe T1R receptors of the invention. Full-length and fragment antisensepolynucleotides are provided. Fragment antisense molecules of theinvention include (i) those that specifically recognize and hybridize toT1R RNA (as determined by sequence comparison of DNA encoding T1Rreceptor to DNA encoding other known molecules). Identification ofsequences unique to T1R-encoding polynucleotides can be deduced throughuse of any publicly available sequence database, and/or through use ofcommercially available sequence comparison programs. Afteridentification of the desired sequences, isolation through restrictiondigestion or amplification using any of the various polymerase chainreaction techniques well known in the art can be performed. Antisensepolynucleotides are particularly relevant to regulation of expression ofT1R receptor by those cells expressing T1R mRNA.

Antisense nucleic acids (preferably 10 to 30 base-pair oligonucleotides)capable of specifically binding to T1R expression control sequences orT1R RNA are introduced into cells (e.g., by a viral vector or colloidaldispersion system such as a liposome). The antisense nucleic acid bindsto the target nucleotide sequence in the cell and prevents transcriptionand/or translation of the target sequence. Phosphorothioate andmethylphosphonate antisense oligonucleotides are specificallycontemplated for therapeutic use by the invention. Locked nucleic acidsare also specifically contemplated for therapeutic use by the presentinvention. (See, for example, Wahlestedt et al., Proc. Natl. Acad. Sci.USA, 97(10), 5633-5638 (2000), which is incorporated by reference in itsentirety) The antisense oligonucleotides may be further modified byadding poly-L-lysine, transferrin polylysine, or cholesterol moieties attheir 5′ end. Suppression of T1R expression at either thetranscriptional or translational level is useful to generate cellular oranimal models for diseases/conditions characterized by aberrant T1Rexpression.

Antisense oligonucleotides to or fragments of nucleotide sequence of SEQID NO:1, SEQ ID NO:99, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:62, or SEQID NO:63, or sequences complementary or homologous thereto, derived fromthe nucleotide sequences of the present invention encoding T1R receptorsare useful as diagnostic tools for probing gene expression in varioustissues. For example, tissue can be probed in situ with oligonucleotideprobes carrying detectable groups by conventional autoradiographytechniques to investigate native expression of this enzyme orpathological conditions relating thereto. Antisense oligonucleotides maybe directed to regulatory regions of a T1R nucleotide sequence, or mRNAcorresponding thereto, including, but not limited to, the initiationcodon, TATA box, enhancer sequences, and the like.

Those of skill in the art recognize that the antisense oligonucleotidesthat inhibit the expression and/or biological activity of a T1R receptorof the invention may be predicted using any gene encoding a T1Rreceptor. Specifically, antisense nucleic acid molecules comprise asequence preferably complementary to at least about 5, 10, 15, 20, 25,30, 35, 40, 45, 50, 100, 250 or 500 nucleotides or an entire T1Rreceptor gene sequence. The antisense oligonucleotides may comprise asequence complementary to about 15 consecutive nucleotides of the codingstrand of the T1R receptor-encoding sequence.

In one embodiment, an antisense nucleic acid molecule is antisense to a“coding region” of the coding strand of a nucleotide sequence encoding aT1R protein. The coding strand may also include regulatory regions ofthe T1R sequence. The term “coding region” refers to the region of thenucleotide sequence comprising codons which are translated into aminoacid residues. In another embodiment, the antisense nucleic acidmolecule is antisense to a “noncoding region” of the coding strand of anucleotide sequence encoding a T1R protein. The term “noncoding region”refers to 5′ and 3′ sequences which flank the coding region that are nottranslated into amino acids (i.e., also referred to as 5′ and 3′untranslated regions (UTR)).

Antisense oligonucleotides may be directed to regulatory regions of anucleotide sequence encoding a T1R protein, or mRNA correspondingthereto, including, but not limited to, the initiation codon, TATA box,enhancer sequences, and the like. Given the coding strand sequencesprovided herein, antisense nucleic acids of the invention can bedesigned according to the rules of Watson and Crick or Hoogsteen basepairing. The antisense nucleic acid molecule can be complementary to theentire coding region of a T1R mRNA, but also may be an oligonucleotidethat is antisense to only a portion of the coding or noncoding region ofthe mRNA. For example, the antisense oligonucleotide can becomplementary to the region surrounding the translation start site of anmRNA. An antisense oligonucleotide can be, for example, about 5, 10, 15,20, 25, 30, 35, 40, 45 or 50 nucleotides in length.

Another means to inhibit the activity of a T1R receptor according to theinvention is via RNA interference (RNAi) (see e.g., Elbashir et al.,Nature, 411:494-498 (2001); Elbashir et al., Genes Development,15:188-200 (2001)). RNAi is the process of sequence-specific,post-transcriptional gene silencing, initiated by double-stranded RNA(dsRNA) that is homologous in sequence to the silenced gene (e.g., ishomologous in sequence to the sequence encoding a T1R receptor, forexample but not limited to the sequence as set forth in SEQ ID NO:1, SEQID NO:99, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:62, or SEQ ID NO:63).siRNA-mediated silencing is thought to occur post-transcriptionallyand/or transcriptionally. For example, siRNA duplexes may mediatepost-transcriptional gene silencing by reconstitution of siRNA-proteincomplexes (siRNPs), which guide mRNA recognition and targeted cleavage.

Accordingly, another form of a T1R inhibitory compound of the inventionis a short interfering RNA (siRNA) directed against a T1R-encodingsequence. Exemplary siRNAs are siRNA duplexes (for example, 10-25,preferably 20, 21, 22, 23, 24, or 25 residues in length) having asequence homologous or identical to a fragment of the T1R sequence setforth as SEQ ID NO:1, SEQ ID NO:99, SEQ ID NO:59, SEQ ID NO:60, SEQ IDNO:62, or SEQ ID NO:63 and having a symmetric 2-nucleotide 3′-overhang.The 2-nucleotide 3′ overhang may be composed of (2′-deoxy) thymidinebecause it reduces costs of RNA synthesis and may enhance nucleaseresistance of siRNAs in the cell culture medium and within transfectedcells. Substitution of uridine by thymidine in the 3′ overhang is alsowell tolerated in mammalian cells, and the sequence of the overhangappears not to contribute to target recognition.

Polypeptides

The invention also provides purified and isolated mammalian (e.g.,feline) T1R receptor polypeptides and dimers. The T1R polypeptides ofthe invention may be encoded by a polynucleotide of the invention. Someembodiments include a feline T1R polypeptide comprising the amino acidsequence of SEQ ID NO:2, SEQ ID NO:61, or SEQ ID NO:64, or fragmentsthereof comprising an epitope specific to the polypeptide. T1R receptorsmay form homodimers or heterodimers in taste buds and other tissues tosense molecules. The invention further comprises homodimeric andheterodimeric forms of T1R receptors wherein one T1R receptor moleculeassociates with a molecule of T1R1, T1R2, or T1R3. A reference to“epitope specific to” or “polypeptide-specific epitope,” or variationsthereof, indicates that a portion of the T1R receptor, T1R receptoramino acid sequence, or T1R dimer is recognizable by an antibody that isspecific for the T1R or amino acid sequence.

Included within the scope of the invention are polypeptides encoded byfeline allelic variants of T1R. The allelic variants of the T1R receptorof the invention may modify the taste perception of a mammal, such as acat, to a taste stimulus. Such functional amino acid sequencemodifications may account for differences in intraspecies (e.g.,breed-specific) taste perception.

Extracellular epitopes are useful for generating and screening forantibodies and other binding compounds that bind to a T1R receptor ordimer. Thus, in another embodiment, the invention provides a purifiedand isolated polypeptide comprising at least one extracellular domain ofthe T1R receptor or dimer. Also included is a polypeptide comprising aT1R receptor fragment selected from the group consisting of anextracellular domain of T1R3 (residues 1-571, 628-641, 705-730, and787-794 of SEQ ID NO:2), a transmembrane domain of T1R3 (residues572-594, 610-627, 642-664, 681-704, 731-754, 767-780, and 795-812 of SEQID NO:2), an intracellular domain of T1R3 (residues 595-609, 665-680,755-766, and 813-865 of SEQ ID NO:2), an extracellular domain of theT1R1 receptor (residues 1-563, 624-635, 701-726, and 781-792 of SEQ IDNO:61), a transmembrane domain of the T1R1 receptor (residues 564-589,604-623, 636-660, 681-700, 727-748, 761-780, and 793-817 of SEQ IDNO:61), an intracellular domain of the T1R1 receptor (residues 590-603,661-680, 749-760, and 818-841 of SEQ ID NO:61), an extracellular domainof T1R2 (residues 1-147 of SEQ ID NO:64), a transmembrane domain of aT1R2 receptor (residues 148-167 of SEQ ID NO:64), and an intracellulardomain of a T1R2 receptor (residues 168-391 of SEQ ID NO:64).Polypeptide fragments of the invention may be continuous portions of thenative receptor. However, it will also be appreciated that knowledge ofthe T1R genes and protein sequences as provided herein permitsrecombination of various domains that are not contiguous in the nativeprotein.

The invention embraces polypeptides that preferably have at least about99%, at least about 95%, at least about 90%, at least about 85%, atleast about 80%, at least about 75%, at least about 74%, at least about73%, at least about 72%, at least about 71%, at least about 70%, atleast about 65%, at least about 60%, at least about 55%, or at leastabout 50% identity and/or homology to the polypeptides of the invention.

Polypeptides of the invention may be isolated from natural cell sourcesor may be chemically synthesized, but are preferably produced byrecombinant procedures involving host cells of the invention. Use ofmammalian host cells is expected to provide for such post-translationalmodifications (e.g., glycosylation, truncation, lipidation, andphosphorylation) as may be needed to confer optimal biological activityon recombinant expression products of the invention.

The invention also embraces variant T1R polypeptides. In one example,insertion variants are provided wherein one or more amino acid residuessupplement a T1R amino acid sequence such as SEQ ID NO:2, SEQ ID NO:61,or SEQ ID NO:64. Insertions may be located at either or both termini ofthe protein, or may be positioned within internal regions of the aminoacid sequence. Insertional variants with additional residues at eitheror both termini can include, for example, fusion proteins and proteinsincluding amino acid tags or labels.

Insertion variants include T1R polypeptides wherein one or more aminoacid residues are added to a biologically active fragment thereof. Forexample, the insertion variants of the invention include chimeric T1Rreceptors wherein at least one functional domain of a feline T1Rreceptor of the invention is present.

The invention also embraces T1R variants having additional amino acidresidues that result from use of specific expression systems. Forexample, use of commercially available vectors that express a desiredpolypeptide as part of a glutathione-S-transferase (GST) fusion productprovides the desired polypeptide having an additional glycine residue atposition −1 after cleavage of the GST component from the desiredpolypeptide. Variants that result from expression in other vectorsystems are also contemplated.

In another aspect, the invention provides deletion variants wherein oneor more amino acid residues in a T1R polypeptide are removed. Deletionscan be effected at one or both termini of the T1R polypeptide, or withremoval of one or more non-terminal amino acid residues of T1R. Deletionvariants, therefore, include all fragments of a T1R polypeptide.

The invention also embraces polypeptide fragments that maintainbiological (e.g., ligand binding, dimerization, receptor activity)and/or immunological properties of a T1R polypeptide.

As used in the present invention, polypeptide fragments preferablycomprise at least 10, 15, 20, 25, 30, 35, 40, 45, or 50 consecutiveamino acids of SEQ ID NO:2, SEQ ID NO:61, or SEQ ID NO:64. Somepolypeptide fragments display antigenic properties unique to, orspecific for, a feline T1R receptor. Fragments of the invention havingthe desired biological and immunological properties can be prepared byany of the methods well known and routinely practiced in the art.

In still another aspect, the invention provides substitution variants ofT1R polypeptides. Substitution variants include those polypeptideswherein one or more amino acid residues of a T1R polypeptide are removedand replaced with alternative residues. In one aspect, the substitutionsare conservative in nature; however, the invention embracessubstitutions that are also non-conservative. Conservative substitutionsfor this purpose may be defined as set out in Tables 1, 2, or 3 below.

Variant polypeptides include those wherein conservative substitutionshave been introduced by modification of polynucleotides encodingpolypeptides of the invention. Amino acids can be classified accordingto physical properties and contribution to secondary and tertiaryprotein structure. A conservative substitution is recognized in the artas a substitution of one amino acid for another amino acid that hassimilar properties. Exemplary conservative substitutions are set out inTable 1 (from WO 97/09433, page 10, published Mar. 13, 1997(PCT/GB96/02197, filed Sep. 6, 1996), immediately below.

TABLE 1 Conservative Substitutions I SIDE CHAIN CHARACTERISTIC AliphaticAMINO ACID Non-polar G A P I L V Polar—uncharged C S T M N QPolar—charged D E K R Aromatic H F W Y Other N Q D EAlternatively, conservative amino acids can be grouped as described inLehninger, [BIOCHEMISTRY, Second Edition; Worth Publishers, Inc. NY,N.Y. (1975), pp. 71-77] as set out in Table 2, below.

TABLE 2 Conservative Substitutions II SIDE CHAIN CHARACTERISTIC AMINOACID Non-polar (hydrophobic) A. Aliphatic: A L I V P B. Aromatic: F W C.Sulfur-containing: M D. Borderline: G Uncharged-polar A. Hydroxyl: S T YB. Amides: N Q C. Sulfhydryl: C D. Borderline: G Positively Charged(Basic): K R H Negatively Charged (Acidic): D EAs still another alternative, exemplary conservative substitutions areset out in Table 3, below.

TABLE 3 Conservative Substitutions III Original Residue ExemplarySubstitution Ala (A) Val, Leu, Ile Arg (R) Lys, Gln, Asn Asn (N) Gln,His, Lys, Arg Asp (D) Glu Cys (C) Ser Gln (Q) Asn Glu (E) Asp His (H)Asn, Gln, Lys, Arg Ile (I) Leu, Val, Met, Ala, Phe, Leu (L) Ile, Val,Met, Ala, Phe Lys (K) Arg, Gln, Asn Met (M) Leu, Phe, Ile Phe (F) Leu,Val, Ile, Ala Pro (P) Gly Ser (S) Thr Thr (T) Ser Trp (W) Tyr Tyr (Y)Trp, Phe, Thr, Ser Val (V) Ile, Leu, Met, Phe, Ala

It should be understood that the definition of polypeptides of theinvention is intended to include polypeptides bearing modificationsother than insertion, deletion, or substitution of amino acid residues.By way of example, the modifications may be covalent in nature, andinclude for example, chemical bonding with polymers, lipids, otherorganic, and inorganic moieties. Such derivatives may be prepared toincrease circulating half-life of a polypeptide, or may be designed toimprove the targeting capacity of the polypeptide for desired cells,tissues, or organs. Similarly, the invention further embraces T1Rpolypeptides that have been covalently modified to include one or morewater-soluble polymer attachments such as polyethylene glycol,polyoxyethylene glycol, or polypropylene glycol. Variants that displayligand binding properties of native T1R and are expressed at higherlevels, as well as variants that provide for constitutively activereceptors, are particularly useful in assays of the invention; thevariants are also useful in providing cellular, tissue and animal modelsof diseases/conditions characterized by aberrant T1R activity.

In a related embodiment, the present invention provides compositionscomprising purified polypeptides of the invention. Some compositionscomprise, in addition to the polypeptide of the invention, apharmaceutically acceptable (i.e., sterile and non-toxic) liquid,semisolid, or solid diluent that serves as a pharmaceutical vehicle,excipient, or medium. Any diluent known in the art may be used.Exemplary diluents include, but are not limited to, water, salinesolutions, polyoxyethylene sorbitan monolaurate, magnesium stearate,methyl- and propylhydroxybenzoate, talc, alginates, starches, lactose,sucrose, dextrose, sorbitol, mannitol, glycerol, calcium phosphate,mineral oil, and cocoa butter.

Variants that display ligand-binding properties of native T1R and areexpressed at higher levels, as well as variants that provide forconstitutively active receptors, are particularly useful in assays ofthe invention; the variants are also useful in assays of the inventionand in providing cellular, tissue and animal models ofdiseases/conditions characterized by aberrant T1R activity.

Antibodies

Also included in the present invention are antibodies (e.g., monoclonaland polyclonal antibodies, single chain antibodies, chimeric antibodies,bifunctional/bispecific antibodies, humanized antibodies, humanantibodies, felinized antibodies, feline antibodies, and complementarydetermining region (CDR)-grafted antibodies, including compounds whichinclude CDR sequences which specifically recognize a polypeptide of theinvention) specific for a T1R receptor of the invention, a fragmentthereof, or dimers of the T1R receptors of the invention (e.g.,T1R1-T1R3; T1R2-T1R3; and T1R3-T1R3 dimers). Antibody fragments,including Fab, Fab′, F(ab′)₂, and F_(v), are also provided by theinvention. The term “specific for,” when used to describe antibodies ofthe invention, indicates that the variable regions of the antibodies ofthe invention recognize and bind T1R polypeptides, preferablyexclusively (i.e., are able to distinguish T1R polypeptides of theinvention from other known polypeptides by virtue of measurabledifferences in binding affinity, despite the possible existence oflocalized sequence identity, homology, or similarity between T1R andsuch polypeptides). It will be understood that specific antibodies mayalso interact with other proteins (for example, S. aureus protein A orother antibodies in ELISA techniques) through interactions withsequences outside the variable region of the antibodies, and, inparticular, in the constant region of the molecule. Screening assays todetermine binding specificity of an antibody of the invention are wellknown and routinely practiced in the art. For a comprehensive discussionof such assays, see Harlow et al. (Eds.), ANTIBODIES A LABORATORYMANUAL; Cold Spring Harbor Laboratory; Cold Spring Harbor, N.Y. (1988),Chapter 6. Antibodies that recognize and bind fragments of the T1Rpolypeptides of the invention are also contemplated, provided that theantibodies are specific for T1R polypeptides. Antibodies of theinvention can be produced using any method well known and routinelypracticed in the art.

The invention provides an antibody that is specific for the feline T1Rreceptors of the invention. Antibodies that can be generated frompolypeptides that have previously been described in the literature andthat are capable of fortuitously cross-reacting with feline T1R receptor(e.g., due to the fortuitous existence of a similar epitope in bothpolypeptides) are considered “cross-reactive” antibodies. Suchcross-reactive antibodies are not antibodies that are “specific” for afeline T1R receptor. The determination of whether an antibody isspecific for a feline T1R receptor or is cross-reactive with anotherknown receptor is made using any of several assays, such as Westernblotting assays, that are well known in the art. For identifying cellsthat express a T1R receptor and also for modulating T1R-ligand bindingactivity, antibodies that specifically bind to an extracellular epitopeof the T1R receptor may be used.

In some embodiments of the invention, the antibodies specifically bindT1R polypeptides, or block ligands or binding partners from binding tothe T1R polypeptides. These antibodies may also block the biologicalactivity of the T1R polypeptides. In other embodiments, the antibodiespreferentially bind T1R polypeptides of a certain species or family ofT1R polypeptides.

In some variations, the invention provides monoclonal antibodies.Hybridomas that produce such antibodies also are intended as aspects ofthe invention. In yet another variation, the invention provides afelinized antibody. Felinized antibodies are useful for in vivotherapeutic indications.

In another variation, the invention provides a cell-free compositioncomprising polyclonal antibodies, wherein at least one of the antibodiesis an antibody of the invention specific for T1R receptor. Antiseraisolated from an animal is an exemplary composition, as is a compositioncomprising an antibody fraction of an antisera that has been resuspendedin water or in another diluent, excipient, or carrier.

In still another related embodiment, the invention provides ananti-idiotypic antibody specific for an antibody that is specific forT1R receptor of the invention.

It is well known that antibodies contain relatively small antigenbinding domains that can be isolated chemically or by recombinanttechniques. Such domains are useful T1R receptor binding moleculesthemselves, and also may be reintroduced into other antibodies or fusedto toxins or other polypeptides. Thus, in still another embodiment, theinvention provides a polypeptide comprising a fragment of a T1R-specificantibody, wherein the fragment and the polypeptide bind to the T1Rreceptor. By way of non-limiting example, the invention providespolypeptides that are single chain antibodies and CDR-graftedantibodies.

Non-feline antibodies may be felinized by any of the methods known inthe art. In one method, the non-feline CDRs are inserted into a felineantibody or consensus antibody framework sequence. Similarly, non-humanantibodies may be humanized by methods known in the art. In oneembodiment, non-human CDRs are inserted into a human antibody orconsensus antibody framework sequence. Further changes can then beintroduced into the antibody framework to modulate affinity orimmunogenicity.

Antibodies of the invention are useful for, e.g., therapeutic purposes(such as by modulating activity of T1R receptor), diagnostic purposes(such as detecting or quantitating T1R receptor activity), and also forpurification of T1R receptor. Kits comprising an antibody of theinvention for any of the purposes described herein are also includedwithin the scope of the invention. In general, a kit of the inventionpreferably includes a control antigen for which the antibody isimmunospecific.

Compositions

Mutations in the feline T1R gene that result in loss of normal functionof the T1R gene product underlie some T1R-related disease states. Theinvention comprehends gene and peptide therapy, for example, to restoreT1R activity to treat those disease states. Delivery of a functional T1Rgene to appropriate cells is effected ex vivo, in situ, or in vivo byuse of vectors, and more particularly viral vectors (e.g., adenovirus,adeno-associated virus, or a retrovirus), or ex vivo by use of physicalDNA transfer methods (e.g., liposomes or chemical treatments). See, forexample, Anderson, Nature, supplement to vol. 392, No. 6679, pp. 25-20(1998). For additional reviews of gene therapy technology see Friedmann,Science, 244: 1275-1281 (1989); Verma, Scientific American: 68-84(1990); and Miller, Nature, 357: 455-460 (1992). Alternatively, it iscontemplated that in other disease states, preventing the expression of,or inhibiting the activity of, T1R receptor will be useful in treatment.It is contemplated that antisense therapy or gene therapy could beapplied to negatively regulate the expression of T1R receptor.

Another aspect of the present invention is directed to compositions,including pharmaceutical compositions, comprising any of the nucleicacid molecules or recombinant expression vectors described above and anacceptable carrier or diluent. The carrier or diluent may bepharmaceutically acceptable. Suitable carriers are described in the mostrecent edition of Remington's Pharmaceutical Sciences, A. Osol, astandard reference text in this field, which is incorporated herein byreference in its entirety. Examples of such carriers or diluentsinclude, but are not limited to, water, saline, Ringer's solution,dextrose solution, and 5% serum albumin. Liposomes and nonaqueousvehicles such as fixed oils may also be used. The formulations may besterilized by commonly used techniques.

Also within the scope of the invention are compositions comprisingpolypeptides, polynucleotides, or antibodies of the invention that havebeen formulated with, e.g., a pharmaceutically acceptable carrier.

The invention also provides methods of using antibodies of theinvention. For example, the invention provides a method for modulatingligand-binding of a T1R receptor comprising the step of contacting thereceptor with an antibody specific for the T1R polypeptide, underconditions wherein the antibody binds the receptor.

Methods of Identifying Ligands and Modulators

The invention also provides assays to identify compounds that bindand/or modulate T1R receptor. A “T1R binding partner” is a compound thatdirectly or indirectly binds a T1R polypeptide of the invention. Oneassay of the invention comprises the steps of: (a) contacting T1Rreceptor or T1R dimer of the invention with a compound suspected ofbinding T1R receptor or dimer (the test compound); and (b) measuringbinding between the compound and the T1R receptor or dimer. In onevariation, the composition comprises a cell expressing T1R receptor ordimer on its surface. In another variation, isolated T1R receptor ordimer or cell membranes comprising T1R receptor or dimer are employed.The binding may be measured directly, e.g., by using a labeled compound,or may be measured indirectly. Compounds identified as binding a T1Rreceptor or T1R dimer may be further tested in other assays including,but not limited to, T1R activity assays and/or in vivo models, in orderto confirm or quantitate their activity.

Specific binding molecules, including natural ligands and syntheticcompounds, can be identified or developed using isolated or recombinantT1R products, T1R variants, or preferably, cells expressing suchproducts. Binding partners are useful for purifying T1R products anddetection or quantification of T1R products in fluid and tissue samplesusing known immunological procedures. Binding molecules are alsomanifestly useful in modulating (i.e., blocking, inhibiting orstimulating) biological activities of T1R, especially those activitiesinvolved in signal transduction. Binding molecules also are useful inmethods for predicting the taste perception of an organism such as amammal by detecting a polypeptide of the invention in a biologicalsample of the organism. For example, an organism in which a polypeptideof the invention has been identified may be attracted to the taste ofamino acids and/or indifferent to the taste of carbohydrate andhigh-intensity sweeteners.

The DNA and amino acid sequence information provided by the presentinvention also makes possible identification of binding partnercompounds with which a T1R polypeptide or polynucleotide will interact.Methods to identify binding partner compounds include solution assays,in vitro assays wherein T1R polypeptides are immobilized, and cell-basedassays. Identification of binding partner compounds of T1R polypeptidesprovides candidates for therapeutic or prophylactic intervention inpathologies associated with T1R normal and aberrant biological activity.

The invention includes several assay systems for identifying T1R-bindingpartners. In solution assays, methods of the invention comprise thesteps of (a) contacting a T1R receptor or dimer with one or morecandidate binding partner compounds and (b) identifying the compoundsthat bind to the T1R receptor or dimer. Identification of the compoundsthat bind the T1R receptor or dimer can be achieved by isolating the T1Rpolypeptide/binding partner complex, and separating the binding partnercompound from the T1R polypeptide. An additional step of characterizingthe physical, biological, and/or biochemical properties of the bindingpartner compound is also comprehended in another embodiment of theinvention. In one aspect, the T1R polypeptide/binding partner complex isisolated using an antibody immunospecific for either the T1R receptor ordimer or the candidate binding partner compound.

In still other embodiments, either the T1R receptor or dimer or thecandidate binding partner compound comprises a label or tag thatfacilitates its isolation, and methods of the invention to identifybinding partner compounds include a step of isolating the T1Rpolypeptide/binding partner complex through interaction with the labelor tag. An exemplary tag of this type is a poly-histidine sequence,generally around six histidine residues, that permits isolation of acompound so labeled using nickel chelation. Other labels and tags, suchas the FLAG® tag (Eastman Kodak, Rochester, N.Y.), well known androutinely used in the art, are embraced by the invention.

In one variation of an in vitro assay, the invention provides a methodcomprising the steps of (a) contacting an immobilized T1R receptor ordimer with a candidate binding partner compound and (b) detectingbinding of the candidate compound to the T1R receptor or dimer. In analternative embodiment, the candidate binding partner compound isimmobilized and binding of T1R receptor or dimer is detected.Immobilization is accomplished using any of the methods well known inthe art, including covalent bonding to a support, a bead, or achromatographic resin, as well as non-covalent, high affinityinteractions such as antibody binding, or use of streptavidin/biotinbinding wherein the immobilized compound includes a biotin moiety. Thesupport may, for example, be formulated into a feline-specificelectronic tongue or biosensor. Detection of binding can be accomplished(i) using a radioactive label on the compound that is not immobilized,(ii) using a fluorescent label on the non-immobilized compound, (iii)using an antibody immunospecific for the non-immobilized compound, (iv)using a label on the non-immobilized compound that excites a fluorescentsupport to which the immobilized compound is attached, as well as othertechniques well known and routinely practiced in the art.

The invention also provides cell-based assays to identify bindingpartner compounds of a T1R receptor or dimer. In one embodiment, theinvention provides a method comprising the steps of contacting a T1Rreceptor or dimer expressed on the surface of a cell with a candidatebinding partner compound and detecting binding of the candidate bindingpartner compound to the T1R receptor or dimer. In some embodiments, thedetection comprises detecting physiological event in the cell caused bythe binding of the molecule.

Another aspect of the present invention is directed to methods ofidentifying compounds that bind to either T1R receptor or dimer ornucleic acid molecules encoding T1R receptor, comprising contacting T1Rreceptor or dimer, or a nucleic acid molecule encoding the same, with acompound, and determining whether the compound binds T1R receptor ordimer or a nucleic acid molecule encoding the same. Binding can bedetermined by binding assays which are well known to the skilledartisan, including, but not limited to, gel-shift assays, Western blots,radiolabeled competition assay, phage-based expression cloning,co-fractionation by chromatography, co-precipitation, cross-linking,interaction trap/two-hybrid analysis, southwestern analysis, ELISA, andthe like, which are described in, for example, CURRENT PROTOCOLS INMOLECULAR BIOLOGY, 1999, John Wiley & Sons, NY, which is incorporatedherein by reference in its entirety. The compounds to be screenedinclude (which may include compounds which are suspected to bind T1Rreceptor or dimer, or a nucleic acid molecule encoding the same), butare not limited to, extracellular, intracellular, biological, orchemical origin. The methods of the invention also embrace ligands thatare attached to a label, such as a radiolabel (e.g., ¹²⁵I, ³⁵S, ³²P,³³P, ³H), a fluorescence label, a chemiluminescent label, an enzymiclabel, and an immunogenic label. Modulators falling within the scope ofthe invention include, but are not limited to, non-peptide moleculessuch as non-peptide mimetics, non-peptide allosteric effectors, andpeptides. The T1R polypeptide or dimer or polynucleotide employed insuch a test may either be free in solution, attached to a solid support,borne on a cell surface or located intracellularly, or associated with aportion of a cell. One skilled in the art can, for example, measure theformation of complexes between T1R receptor, dimer, or polynucleotideand the compound being tested. Alternatively, one skilled in the art canexamine the diminution in complex formation between T1R receptor, dimer,or polynucleotide and its substrate caused by the compound being tested.In some embodiments of the invention, the recognition sites of the T1Rreceptor, dimer, or polypeptide are coupled with a monitoring system,either electrical or optical. An appropriate chemical stimulus can bindto the receptor's ligand binding domain, changing the receptorconformation to a degree that the coupled electronics or optical changescan be observed on a read-out. Such a device could be developed into afeline-specific electronic tongue, for example.

In another embodiment of the invention, high throughput screening forcompounds having suitable binding affinity to T1R receptor or dimer isemployed. Briefly, large numbers of different small peptide testcompounds are synthesized on a solid substrate. The peptide testcompounds are contacted with T1R receptor or dimer and washed. Bound T1Rreceptor or dimer is then detected by methods well known in the art.Purified polypeptides of the invention can also be coated directly ontoplates for use in the aforementioned drug screening techniques. Inaddition, non-neutralizing antibodies can be used to capture the proteinand immobilize it on the solid support.

Generally, an expressed T1R receptor can be used for HTS binding assaysin conjunction with a ligand, such as an amino acid or carbohydrate. Theidentified peptide is labeled with a suitable radioisotope, including,but not limited to, ¹²⁵I, ³H, ³⁵S, or ³²P, by methods that are wellknown to those skilled in the art. Alternatively, the peptides may belabeled by well-known methods with a suitable fluorescent derivative(Baindur et al., Drug Dev. Res., 1994, 33, 373-398; Rogers, DrugDiscovery Today, 1997, 2, 156-160). Radioactive ligand specificallybound to the receptor in membrane preparations made from the cell lineexpressing the recombinant protein can be detected in HTS assays in oneof several standard ways, including filtration of the receptor-ligandcomplex to separate bound ligand from unbound ligand (Williams, Med.Res. Rev., 1991, 11, 147-184; Sweetnam et al., J. Natural Products,1993, 56, 441-455). Alternative methods include a scintillationproximity assay (SPA) or a FlashPlate format in which such separation isunnecessary (Nakayama, Cur. Opinion Drug Disc. Dev., 1998, 1, 85-91;Bossé et al., J. Biomolecular Screening, 1998, 3, 285-292.). Binding offluorescent ligands can be detected in various ways, includingfluorescence energy transfer (FRET), direct spectrophotofluorometricanalysis of bound ligand, or fluorescence polarization (Rogers, DrugDiscovery Today, 1997, 2, 156-160; Hill, Cur. Opinion Drug Disc. Dev.,1998, 1, 92-97).

Other assays may be used to identify specific ligands of a T1R receptoror dimer, including assays that identify ligands of the target proteinthrough measuring direct binding of test ligands to the target, as wellas assays that identify ligands of target proteins through affinityultrafiltration with ion spray mass spectroscopy/HPLC methods or otherphysical and analytical methods. Alternatively, such bindinginteractions are evaluated indirectly using the yeast two-hybrid systemdescribed in Fields et al., Nature, 340:245-246 (1989), and Fields etal., Trends in Genetics, 10:286-292 (1994), both of which areincorporated herein by reference. The two-hybrid system is a geneticassay for detecting interactions between two proteins or polypeptides.It can be used to identify proteins that bind to a known protein ofinterest, or to delineate domains or residues critical for aninteraction. Variations on this methodology have been developed to clonegenes that encode DNA binding proteins, to identify peptides that bindto a protein, and to screen for drugs. The two-hybrid system exploitsthe ability of a pair of interacting proteins to bring a transcriptionactivation domain into close proximity with a DNA binding domain thatbinds to an upstream activation sequence (UAS) of a reporter gene, andis generally performed in yeast. The assay requires the construction oftwo hybrid genes encoding (1) a DNA-binding domain that is fused to afirst protein and (2) an activation domain fused to a second protein.The DNA-binding domain targets the first hybrid protein to the UAS ofthe reporter gene; however, because most proteins lack an activationdomain, this DNA-binding hybrid protein does not activate transcriptionof the reporter gene. The second hybrid protein, which contains theactivation domain, cannot by itself activate expression of the reportergene because it does not bind the UAS. However, when both hybridproteins are present, the noncovalent interaction of the first andsecond proteins tethers the activation domain to the UAS, activatingtranscription of the reporter gene. For example, when the first proteinis a receptor, or fragment thereof, that is known to interact withanother protein or nucleic acid, this assay can be used to detect agentsthat interfere with the binding interaction. Expression of the reportergene is monitored as different test agents are added to the system. Thepresence of an inhibitory agent results in lack of a reporter signal.

The yeast two-hybrid assay can also be used to identify proteins thatbind to the gene product. In an assay to identify proteins that bind toa T1R receptor, or fragment thereof, a fusion polynucleotide encodingboth a T1R receptor (or fragment) and a UAS binding domain (i.e., afirst protein) may be used. In addition, a large number of hybrid geneseach encoding a different second protein fused to an activation domainare produced and screened in the assay. Typically, the second protein isencoded by one or more members of a total cDNA or genomic DNA fusionlibrary, with each second protein-coding region being fused to theactivation domain. This system is applicable to a wide variety ofproteins, and it is not necessary to know the identity or function ofthe second binding protein. The system is highly sensitive and candetect interactions not revealed by other methods; even transientinteractions may trigger transcription to produce a stable mRNA that canbe repeatedly translated to yield the reporter protein.

Other assays may be used to search for agents that bind to the targetprotein. One such screening method to identify direct binding of testligands to a target protein is described in U.S. Pat. No. 5,585,277,incorporated herein by reference. This method relies on the principlethat proteins generally exist as a mixture of folded and unfoldedstates, and continually alternate between the two states. When a testligand binds to the folded form of a target protein (i.e., when the testligand is a ligand of the target protein), the target protein moleculebound by the ligand remains in its folded state. Thus, the folded targetprotein is present to a greater extent in the presence of a test ligandwhich binds the target protein, than in the absence of a ligand. Bindingof the ligand to the target protein can be determined by any method thatdistinguishes between the folded and unfolded states of the targetprotein. The function of the target protein need not be known in orderfor this assay to be performed. Virtually any agent can be assessed bythis method as a test ligand, including, but not limited to, metals,polypeptides, proteins, lipids, polysaccharides, polynucleotides andsmall organic molecules.

Another method for identifying ligands of a target protein is describedin Wieboldt et al., Anal. Chem., 69:1683-1691 (1997), incorporatedherein by reference. This technique screens combinatorial libraries of20-30 agents at a time in solution phase for binding to the targetprotein. Agents that bind to the target protein are separated from otherlibrary components by simple membrane washing. The specifically selectedmolecules that are retained on the filter are subsequently liberatedfrom the target protein and analyzed by HPLC and pneumatically assistedelectrospray (ion spray) ionization mass spectroscopy. This procedureselects library components with the greatest affinity for the targetprotein, and is particularly useful for small molecule libraries.

Other embodiments of the invention comprise using competitive screeningassays in which neutralizing antibodies capable of binding a polypeptideof the invention specifically compete with a test compound for bindingto the polypeptide. In this manner, the antibodies can be used to detectthe presence of any peptide that shares one or more antigenicdeterminants with T1R receptor. Radiolabeled competitive binding studiesare described in A. H. Lin et al., Antimicrobial Agents andChemotherapy, 1997, 41(10): 2127-2131, the disclosure of which isincorporated herein by reference in its entirety.

Another aspect of the present invention is directed to methods ofidentifying compounds that modulate (i.e., increase or decrease)activity of T1R receptor or dimer comprising contacting T1R receptor ordimer with a compound, and determining whether the compound modifiesactivity of T1R receptor or dimer. The activity in the presence of thetest compound is compared to the activity in the absence of the testcompound. Where the activity of the sample containing the test compoundis higher than the activity in the sample lacking the test compound, thecompound is an agonist. Similarly, where the activity of the samplecontaining the test compound is lower than the activity in the samplelacking the test compound, the compound is an antagonist.

Agents that modulate (i.e., increase, decrease, or block) T1R receptoror dimer activity or expression also may be identified, for example, byincubating a putative modulator with a cell containing a T1Rpolypeptide, dimer, or polynucleotide and determining the effect of theputative modulator on T1R receptor activity or expression. Theselectivity of a compound that modulates the activity of T1R receptor ordimer can be evaluated by comparing its effects on T1R receptor or dimerto its effect on other T1R receptors or dimers. Selective modulators mayinclude, for example, antibodies and other proteins, peptides, ororganic molecules that specifically bind to a T1R polypeptide or a T1Rreceptor-encoding nucleic acid. Modulators of T1R receptor activity willbe therapeutically useful in treatment of diseases and physiologicalconditions in which normal or aberrant T1R receptor activity isinvolved. Compounds identified as modulating T1R receptor activity maybe further tested in other assays including, but not limited to, in vivomodels, in order to confirm or quantitate their activity.

The invention also provides methods for identifying a T1R receptormodulator by: (a) contacting a T1R receptor (or dimer) binding partnerand a composition comprising a T1R receptor in the presence and in theabsence of a putative modulator compound; (b) detecting binding betweenthe binding partner and the T1R receptor; and (c) identifying a putativemodulator compound or a modulator compound in view of decreased orincreased binding between the binding partner and the T1R receptor inthe presence of the putative modulator, as compared to binding in theabsence of the putative modulator. Compounds identified as modulators ofbinding between T1R receptor and a T1R binding partner may be furthertested in other assays including, but not limited to, in vivo models, inorder to confirm or quantitate their activity.

The invention also includes within its scope high-throughput screening(HTS) assays to identify compounds that interact with, enhance, orinhibit biological activity (i.e., affect enzymatic activity, bindingactivity, etc.) of a T1R receptor or dimer. HTS assays permit screeningof large numbers of compounds in an efficient manner. Cell-based HTSsystems are contemplated to investigate T1R receptor-ligand interaction.HTS assays are designed to identify “hits” or “lead compounds” havingthe desired property, from which modifications can be designed toimprove the desired property. Chemical modification of the “hit” or“lead compound” is often based on an identifiable structure/activityrelationship between the “hit” and the T1R polypeptide.

For example, modulators of T1R receptor activity may be identified byexpressing the T1R receptor in a heterologous cultured mammalian cellline, such as HEK cells, and detecting receptor activity in the presenceand absence of a test compound by monitoring changes in intracellularcalcium using a calcium-specific intracellular dye. In anotherembodiment, this process may be automated using a high-throughputscreening device.

Candidate modulators contemplated by the invention include compoundsselected from libraries of either potential activators or potentialinhibitors. There are a number of different libraries used for theidentification of small molecule modulators, including: (1) chemicallibraries, (2) natural product libraries, and (3) combinatoriallibraries comprised of random peptides, oligonucleotides, or organicmolecules. Chemical libraries consist of random chemical structures,some of which are analogs of known compounds or analogs of compoundsthat have been identified as “hits” or “leads” in other drug discoveryscreens, some of which are derived from natural products, and some ofwhich arise from non-directed synthetic organic chemistry. Naturalproduct libraries are collections of microorganisms, animals, plants, ormarine organisms that are used to create mixtures for screening by: (1)fermentation and extraction of broths from soil, plant, or marinemicroorganisms or (2) extraction of plants or marine organisms. Naturalproduct libraries include polyketides, non-ribosomal peptides, andvariants (non-naturally occurring) thereof. For a review, see Science282:63-68 (1998). Combinatorial libraries are composed of large numbersof peptides, oligonucleotides, or organic compounds as a mixture. Theselibraries are relatively easy to prepare by traditional automatedsynthesis methods, PCR, cloning, or proprietary synthetic methods. Ofparticular interest are non-peptide combinatorial libraries. Still otherlibraries of interest include peptide, protein, peptidomimetic,multiparallel synthetic collection, recombinatorial, and polypeptidelibraries. For a review of combinatorial chemistry and libraries createdtherefrom, see Myers, Curr. Opin. Biotechnol. 8:701-707 (1997).Identification of modulators through use of the various librariesdescribed herein permits modification of the candidate “hit” (or “lead”)to optimize the capacity of the “hit” to modulate activity.

T1R receptor binding partners that stimulate T1R receptor activity areuseful as agonists in disease states or conditions characterized byinsufficient T1R receptor signaling (e.g., as a result of insufficientactivity of a T1R receptor ligand). T1R receptor binding partners thatblock ligand-mediated T1R receptor signaling are useful as T1R receptorantagonists to treat disease states or conditions characterized byexcessive T1R receptor signaling. Thus, in another aspect, the inventionprovides methods for treating a disease or abnormal condition byadministering to a patient in need of such treatment a substance thatmodulates the activity or expression of a polypeptide having a sequenceof SEQ ID NO:2, SEQ ID NO:61, or SEQ ID NO:64, or exhibitingsubstantially the same biological activity as a polypeptide having asequence of SEQ ID NO:2, SEQ ID NO:61, or SEQ ID NO:64, or dimersthereof.

In addition T1R receptor modulators in general, as well as T1R receptorencoding polynucleotides and polypeptides, are useful in diagnosticassays for such diseases or conditions.

The T1R polynucleotides of the invention may also be used to identifycompounds for which an organism having the receptor exhibits a tastepreference or indifference using cell signaling assays known in the art.In such assays, polynucleotide encoding a T1R is incorporated into anexpression vector and transfected into a host cell. The expression ofthe T1R may be inducible or constitutive. The host cells expressing theT1R receptor are contacted with candidate compounds and the effect ofeach compound on the cells is assayed. Stimulation of a response isindicative of reactivity to the test compound and correlates withcompounds associated with a taste preference. The assays that can beused to assess stimulation of T1R receptor include, but are not limitedto assays measuring ion conductance, ion flow, calcium imaging (e.g.,using fura-2, green dextran activity or aequorin activity), voltagemeasurement and or voltage imaging with dyes, expression of reportergenes (e.g., luciferase, alkaline phosphatase, beta-galactosidase,beta-lactamase, fluorescent binding protein), receptor binding assays,second messenger assays (e.g., IP3, cAMP), G-protein activation basedassays (e.g., modulation of GTP-gamma-S binding), receptorphosphorylation measures, and the like. In some embodiments analysis ofstimulation of T1R receptors and receptor dimers may be determined usinga FLIPR® assay as described by the manufacturer (Molecular Devices,Corp.).

Screening of cells treated with dyes and fluorescent reagents is wellknown in the art. Genetic engineering of cells to produce fluorescentproteins, such as modified green fluorescent protein (GFP), as areporter molecule is also well known in the art. Fluorescence-basedreagents are useful for the assay of many cell functions including ionconcentrations, membrane potential, specific translocations, enzymeactivities, gene expression, as well as the presence, amounts andpatterns of metabolites, proteins, lipids, carbohydrates, and nucleicacid sequences.

Cell signaling assays known in the art may also be used to identify T1Rantagonists. Expression of G protein coupled receptors at very highconcentration in a heterologous system has been shown to result inconstitutive cell signaling. For example, but not by way of limitation,T1R receptor may be overexpressed in Spodoptera frugiperda (Sf9) cells.Alternatively, for example, T1R may be operably linked to a CMV promoterand expressed in COS or HEK293 cells. In the activated constitutivestate, test compounds may be assayed for their ability to inhibitconstitutive cell signaling activity. Suitable assays include, but arenot limited to assays measuring ion conductance, ion flow, calciumimaging (e.g., using fura-2, green dextran activity or aequorinactivity), voltage measurement and/or voltage imaging with dyes,expression of reporter genes (e.g., luciferase, alkaline phosphatase,beta-galactosidase, beta-lactamase, fluorescent binding protein),receptor binding assays, second messenger assays (e.g., IP3, cAMP),G-protein activation based assays (e.g., modulation of GTP-gamma-Sbinding), receptor phosphorylation measures, and the like.

Tissue Distribution

One skilled in the art may determine whether a T1R receptor is expressedin tissues other than taste bud tissues through routine experimentationfollowing the guidance provided herein. Specifically, one may detect T1RRNA using Northern blots or reverse transcriptase polymerase chainreaction (rtPCR) and other methods known in the art. Protocols andprocedures for such assays may be found, for example in Ausubel et al.,CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York,1998; Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2D ED.,Cold Spring Harbor Laboratory Press, Plainview, N.Y., 1989.

One may also detect T1R protein using specific antibodies and probingtissue sections. For example, but not by way of limitation, one mayobtain paraffin-embedded sections of tissues of interest (from human orother species) or use fresh tissue. Tissue sections may be treated with0.3% H₂O₂ in methanol for 30 minutes to eliminate endogenous peroxidaseactivity. The tissues may then be incubated in anti-T1R antibody (or anantisera raised against T1R) at 4° C. for hours to days. The tissues maythen be treated with a secondary antibody that specifically binds to theprimary antibody (such as a heterologous species' serum raised againstthe primary species immunoglobulin). The secondary antibody may beconjugated to biotin, for example. The tissue sections are furtherincubated with peroxidase-conjugated streptavidin at room temperature.After each step, tissue sections are rinsed in 0.01 M phosphate bufferedsaline (pH 7.2) containing 0.05% Tween 20 Immunoreaction products may bevisualized through reaction with 0.0125% diaminobenzidine (DAB) and0.002% H₂O₂ in 0.05M Tris-HCl buffer (pH 7.6). The sections arecounterstained with hematoxylin and viewed with a light microscope.Other antibody-based assays to examine tissues for T1R expression may beused. Various protocols are known in the art and may be found, forexample, in Harlow et al. (Eds.), ANTIBODIES A LABORATORY MANUAL; ColdSpring Harbor Laboratory; Cold Spring Harbor, N.Y. (1988).

The presence of T1R receptor in tissues outside of taste buds suggests arole for T1R receptors other than taste perception. The inventionprovides methods for modulating these functions by increasing ordecreasing the expression of T1R.

As such, the invention encompasses a method of modulating a T1R receptorfamily member by modulating expression of another T1R family receptor.For example, the Tas1r gene may be expressed such that an increasedlevel of Tas1r RNA results in feedback inhibition of expression of atleast one of T1R1, T1R2, and T1R3. In other embodiments of theinvention, the Tas1r gene may be expressed such that an increased levelof T1R RNA results in an increase in the expression of at least one ofT1R1, T1R2, and T1R3. In other embodiments, the Tas1r gene may berepressed such that a decreased level of Tas1r RNA results in anincrease in the expression of at least one of T1R1, T1R2, and T1R3. Inother embodiments, the Tas1r gene may be repressed such that a decreasedlevel of Tas1r RNA results in a concomitant decrease in the expressionof at least one of T1R1, T1R2, and T1R3. The gene modulation may betissue- or species-specific. Thus, expression of Tas1r may be regulatedusing the expression vectors of the invention using inducible promotersand tissue-specific promoters, for example. Alternatively, expression ofTas1r may be repressed in cells that produce T1R using antisense RNA andother inhibitory strategies described herein and as are known in theart. The modulation of the T1R receptor family in patients may be usefulfor altering metabolism, nutrient uptake, neural function, and to treatdisorders associated with an abnormally low or high level of T1Rexpression in cells.

Mimetics

Mimetics or mimics of compounds identified herein (sterically similarcompounds formulated to mimic the key portions of the structure) may bedesigned for pharmaceutical use. Mimetics may be used in the same manneras the compounds identified by the present invention that modulate theT1R receptor and hence are also functional equivalents. The generationof a structural-functional equivalent may be achieved by the techniquesof modeling and chemical design known to those of skill in the art. Itwill be understood that all such sterically similar constructs fallwithin the scope of the present invention.

The design of mimetics to a known pharmaceutically active compound is aknown approach to the development of pharmaceuticals based on a “lead”compound. This is desirable where, for example, the active compound isdifficult or expensive to synthesize, or where it is unsuitable for aparticular method of administration, e.g., some peptides may beunsuitable active agents for oral compositions as they tend to bequickly degraded by proteases in the alimentary canal.

There are several steps commonly taken in the design of a mimetic.First, the particular parts of the compound that are critical and/orimportant in determining its T1R-modulating properties are determined.In the case of a polypeptide, this can be done by systematically varyingthe amino acid residues in the peptide, e.g. by substituting eachresidue in turn. Alanine scans of peptides are commonly used to refinesuch peptide motifs.

Once the active region of the compound has been identified, itsstructure is modeled according to its physical properties, e.g.stereochemistry, bonding, size, and/or charge, using data from a rangeof sources, such as, but not limited to, spectroscopic techniques, X-raydiffraction data, and NMR. Computational analysis, similarity mapping(which models the charge and/or volume of the active region, rather thanthe bonding between atoms), and other techniques known to those of skillin the art can be used in this modeling process.

In a variant of this approach, the three-dimensional structure of thecompound that modulates a T1R receptor and the active region of the T1Rreceptor are modeled. This can be especially useful where either or bothof these compounds change conformation upon binding. Knowledge of thestructure of the ligand-binding domain (for example, residues 1-571 ofSEQ ID NO:2) of the receptor also allows the design of high potencyligands and/or modulators.

A template molecule is then selected onto which chemical groups thatmimic the T1R modulator can be grafted. The template molecule and thechemical groups grafted onto it can conveniently be selected so that themimetic is easy to synthesize, is pharmacologically acceptable, and doesnot degrade in vivo, while retaining the biological activity of the leadcompound. Alternatively, where the mimetic is peptide-based, furtherstability can be achieved by cyclizing the peptide, thereby increasingits rigidity. The mimetic or mimetics found by this approach can then bescreened by the methods of the present invention to see whether theyhave the ability to modulate the T1R receptor. Further optimization ormodification can then be performed to arrive at one or more finalmimetics for in vivo or clinical testing.

Compositions of Binding and/or Modulating Compounds

Following identification of a compound that binds and/or or modulates aT1R receptor, the compound may be manufactured and/or used inpreparation of compositions including, but not limited to, foods,drinks, and pharmaceutical compositions. The compositions are providedor administered to patients, including, but not limited to, avians,felines, canines, bovines, ovines, porcines, equines, rodents, simians,and humans.

Thus, the present invention extends, in various aspects, not only tocompounds identified in accordance with the methods disclosed herein butalso foods, drinks, pharmaceutical compositions, drugs, or othercompositions comprising such a compound; methods comprisingadministration of such a composition to a patient, e.g. for treatment(which includes prophylactic treatment) of a T1R receptor-associateddisorder (e.g., obesity, diabetes); uses of such a compound in themanufacture of a composition for administration to a patient; andmethods of making a composition comprising admixing such a compound witha pharmaceutically acceptable excipient, vehicle or carrier, andoptionally other ingredients.

Some compositions of the invention comprise a taste-modifying amount ofat least one or more binding or modulating compounds. A “taste-modifyingamount” is a quantity sufficient to increase or decrease the perceptionof a taste stimulus by a given mammal. The food and drink compositionsof the invention are formulated by the addition of a binding ormodulating compound to a food or drink of the mammal. Such compositionsmay be individualized or breed-specific. For example, feline veterinaryspecialty diets may thus be made more palatable.

The pharmaceutical compositions of the invention comprise atherapeutically effective amount of a compound identified according tothe methods disclosed herein, or a pharmaceutically acceptable saltthereof, and a pharmaceutically acceptable carrier or excipient.

The compounds of the invention can be formulated as neutral or saltforms. Pharmaceutically acceptable salts include those formed with freeamino groups such as those derived from hydrochloric, phosphoric,acetic, oxalic, tartaric acids, etc., and those formed with freecarboxyl groups such as those derived from sodium, potassium, ammonium,calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

Pharmaceutically acceptable carriers include but are not limited tosaline, buffered saline, dextrose, water, glycerol, ethanol, andcombinations thereof. The carrier and composition can be sterile. Theformulation should suit the mode of administration.

The composition, if desired, can also contain minor amounts of wettingor emulsifying agents or pH buffering agents. The composition can be aliquid solution, suspension, emulsion, tablet, pill, capsule, sustainedrelease formulation, or powder. The composition can be formulated as asuppository, with traditional binders and carriers such astriglycerides. Oral formulations can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc.

The pharmaceutical compositions of the invention may further comprise asecondary compound for the treatment of a disorder unrelated to the T1Rreceptor, such as an antibiotic or other therapeutic agent, to improvethe palatability of the pharmaceutical composition, thereby improvingthe ease of administration.

In one embodiment, the composition is formulated in accordance withroutine procedures as a pharmaceutical composition adapted for oral(e.g., tablets, granules, syrups) or non-oral (e.g., ointments,injections) administration to the subject. Various delivery systems areknown and can be used to administer a compound that modulates a T1Rreceptor, e.g., encapsulation in liposomes, microparticles,microcapsules, expression by recombinant cells, receptor-mediatedendocytosis, construction of a therapeutic nucleic acid as part of aretroviral or other vector, etc. Methods of introduction include but arenot limited to intradermal, intramuscular, intraperitoneal, intravenous,subcutaneous, intranasal, topical, and oral routes.

The compounds of the invention may be administered by any convenientroute, for example by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.), and may be administered together with otherbiologically active agents, for example in HAART therapy. Administrationcan be systemic or local. In addition, it may be desirable to introducethe pharmaceutical compositions of the invention into the centralnervous system by any suitable route, including intraventricular andintrathecal injection; intraventricular injection may be facilitated byan intraventricular catheter, for example, attached to a reservoir, suchas an Ommaya reservoir.

In a specific embodiment, it may be desirable to administer thepharmaceutical compositions of the invention locally to the area in needof treatment; this may be achieved by, for example, and not by way oflimitation, local infusion during surgery; topical application, e.g., inconjunction with a wound dressing after surgery; by injection; by meansof a catheter; by means of a suppository; or by means of an implant,said implant being of a porous, non-porous, or gelatinous material,including membranes, such as sialastic membranes, or fibers.

The composition can be administered in unit dosage form and may beprepared by any of the methods well known in the pharmaceutical art, forexample, as described in REMINGTON′S PHARMACEUTICAL SCIENCES (MackPublishing Co., Easton, Pa.). The amount of the compound of theinvention that modulates a T1R receptor that is effective in thetreatment of a particular disorder or condition will depend on factorsincluding but not limited to the chemical characteristics of thecompounds employed, the route of administration, the age, body weight,and symptoms of a patient, the nature of the disorder or condition, andcan be determined by standard clinical techniques. Typically therapy isinitiated at low levels of the compound and is increased until thedesired therapeutic effect is achieved. In addition, in vitro assays mayoptionally be employed to help identify optimal dosage ranges. Suitabledosage ranges for intravenous administration are preferably generallyabout 20-500 micrograms of active compound per kilogram body weight.Suitable dosage ranges for intranasal administration are preferablygenerally about 0.01 pg/kg body weight to 1 mg/kg body weight.Suppositories preferably generally contain active ingredient in therange of 0.5% to 10% by weight; oral formulations preferably may contain10% to 95% active ingredient. Effective doses may be extrapolated fromdose-response curves derived from in vitro or animal model test systems.

Typically, compositions for intravenous administration are solutions insterile isotonic aqueous buffer. Where necessary, the composition mayalso include a solubilizing agent and a local anesthetic such aslidocaine to ease pain at the site of the injection. Generally, theingredients are supplied either separately or mixed together in unitdosage form, for example, as a dry-lyophilized powder or water-freeconcentrate in a hermetically sealed container such as an ampoule orsachette indicating the quantity of active agent. Where the compositionis to be administered by infusion, it can be dispensed with an infusionbottle containing sterile pharmaceutical grade water or saline.

Where the composition is administered by injection, an ampoule ofsterile water for injection or saline can be provided so that theingredients may be mixed prior to administration.

Methods for Predicting and Representing Taste Perception of an Organism

Methods for predicting the taste perception of an organism, such as amammal, include detection of a nucleotide sequence (e.g., nucleotidesequence of SEQ ID NO:1, SEQ ID NO:99, SEQ ID NO:59; SEQ ID NO:60, SEQID NO:62, or SEQ ID NO:63, or fragments, variants, or homologues thereofencoding a polypeptide having an equivalent, substantially the same, orsame biological activity as the polypeptide encoded by the referencesequence) or amino acid sequence (e.g., SEQ ID NO:2, SEQ ID NO:61, orSEQ ID NO:64, or fragments, variants, or homologues thereof having thesame or substantially the same biological activity of the referencepolypeptide) of the invention in a biological sample of the organism.Methods for detecting a polynucleotide or polypeptide of the inventionin a biological sample may comprise any method known to the skilledartisan, including but not limited to the methods of detection mentionedherein. An organism having a polynucleotide or polypeptide of theinvention in its biological sample is predicted to exhibitcharacteristics of taste perception of a domestic cat. For example, anorganism having a polynucleotide or polypeptide of the invention, suchas a polynucleotide encoding a feline T1R1 receptor or the polypeptideencoded thereby, will be attracted to one or more amino acids. Anorganism having a polynucleotide or polypeptide of the invention mayshow no preference for one or more carbohydrates or high-intensitysweeteners. As an example, an organism having a polynucleotide encodinga single transmembrane domain T1R2 receptor, for example the feline T1R2receptor of the invention, or the polypeptide encoded thereby, would bepredicted to show a reduced or no response to sweet taste stimulirelative to an organism having a 7-transmembrane domain T1R2 receptor Anorganism having a polynucleotide or polypeptide of the invention may beattracted to one or more amino acids and may exhibit no preference forone or more carbohydrates or high-intensity sweeteners. For example, anorganism having a polynucleotide or polypeptide of the invention, suchas a polynucleotide encoding a feline T1R1 receptor or the polypeptideencoded thereby, and further having a polynucleotide encoding a singletransmembrane domain T1R2 receptor, for example the feline T1R2 receptorof the invention, or the polypeptide encoded thereby, will be attractedto one or more amino acids and will exhibit no preference for one ormore carbohydrates or high-intensity sweeteners.

Also contemplated by the invention is a method for representing tasteperception of a particular taste in a mammal, for example a cat, byproviding values X₁ to X_(n) representative of the quantitativestimulation of each of n T1R receptors or dimers of the invention, wheren is greater than or equal to 2, and generating from the values aquantitative representation of taste perception of the mammal. Therepresentation may constitute a point or a volume in n-dimensionalspace, may constitute a graph or a spectrum, and/or may constitute amatrix of quantitative representations. Also, the providing step maycomprise contacting a plurality of T1R receptors or dimers of theinvention with a test compound and quantitatively measuring theinteraction of the compound with T1R receptors or dimers.

Also contemplated by the invention are methods for predicting the tasteperception in a mammal generated by one or more molecules orcombinations of molecules yielding unknown taste perception in a mammal,such as a cat, by providing values X₁ to X_(n) representative of thequantitative stimulation of each of n T1R receptors of the invention,where n is greater than or equal to 2, for one or more molecules orcombinations of molecules yielding known taste perception in a mammal;and generating from the values a quantitative representation of tasteperception in a mammal for the one or more molecules or combinations ofmolecules yielding known taste perception in a mammal, providing valuesX₁ to X_(n) representative of the quantitative stimulation of each of nT1R receptors of the invention, where n is greater than or equal to 2,for one or more molecules or combinations of molecules yielding unknowntaste perception in a mammal; and generating from said values aquantitative representation of taste perception in a mammal for the oneor more molecules or combinations of molecules yielding unknown tasteperception in a mammal, and predicting the taste perception in a mammalgenerated by one or more molecules or combinations of molecules yieldingunknown taste perception in a mammal by comparing the quantitativerepresentation of taste perception in a mammal for the one or moremolecules or combinations of molecules yielding unknown taste perceptionin a mammal to the quantitative representation of taste perception in amammal for the one or more molecules or combinations of moleculesyielding known taste perception in a mammal.

In another embodiment, novel molecules or combinations of molecules aregenerated which elicit a predetermined taste perception in a mammal bydetermining a value of taste perception in a mammal such as a cat for aknown molecule or combinations of molecules as described above;determining a value of taste perception in a mammal for one or moreunknown molecules or combinations of molecules as described above;comparing the value of taste perception in a mammal for one or moreunknown compositions to the value of taste perception in a mammal forone or more known compositions; selecting a molecule or combination ofmolecules that elicits a predetermined taste perception in a mammal; andcombining two or more unknown molecules or combinations of molecules toform a molecule or combination of molecules that elicits a predeterminedtaste perception in a mammal. The combining step yields a singlemolecule or a combination of molecules that elicits a predeterminedtaste perception in a mammal.

Treatment Methods

The invention provides methods of treatment of T1R receptor-associateddisorders by administering to a subject or patient an effective amountof a compound that modulates the T1R receptor. In some aspects of theinvention, the compounds or pharmaceutical compositions of the inventionare administered to a patient having an increased risk of or having adisorder associated with the T1R receptor. The patient may be, forexample, avian, feline, canine, bovine, ovine, porcine, equine, rodent,simian, or human.

Kits

A kit of the invention comprises a carrier means being compartmentalizedto receive in close confinement one or more container means such asvials, tubes, and the like, each of the container means comprising anelement to be used in the methods of the invention. For example, one ofthe container means may comprise the a polynucleotide encoding a T1Rreceptor of the invention, a T1R receptor of the invention, or anantibody thereto. The kit may also have one or more conventional kitcomponents, including, but not limited to, instructions, test tubes,Eppendorf™ tubes, labels, reagents helpful for quantification of markergene expression, etc.

EXAMPLES

The following examples are meant to be illustrative of the presentinvention and are not intended to limit the scope thereof.

Cloning and Characterization of the Feline T1R3 Receptor

The discovery of feline taste receptor, T1R3, was achieved by using amolecular strategy termed “overgo” (Thomas, et al., Genome Res.,12:1277-1285 (2002); Vollrath, D., DNA markers for physical mapping InGENOME ANALYSIS: A LABORATORY MANUAL, Vol. 4, ed. B. Birren, et al., pp.187-215, 1999). Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y.). This strategy involves the use of the shortest DNA probes amongthe many kinds of probes used in bacterial artificial chromosome (BAC)library screening. These probes are comprised of two DNA sequences(e.g., 22mers) with a complementary 8 base overlap. They can be designedby computer program available online, for example, through theWashington University School of Medicine Genome Sequencing Center, andare readily synthesized.

Overgo probes were designed from conserved regions of the chromosome 1marker, “disheveled 1” (DVL1) and the G protein-coupled receptor, T1R3,by aligning DVL1 and T1R3 genomic sequences from several species. Theoverlapping sequences of the seven DVL1 overgo probes used in thepresent invention were as follows:

catOV1a (SEQ ID NO: 21) ACTTTGAGAACATGAGTAATGACG catOV1b (SEQ ID NO: 22)AGTACCCGGACTGCGTCGTCATTA catOV2a (SEQ ID NO: 23)CACTAGGGTCATCCTTGCTTTCAG catOV2b (SEQ ID NO: 24)AGTCAGGGTGATGGGCCTGAAAGC Ov8-OVa (SEQ ID NO: 25)ATGTGGTGGACTGGCTGTACCATC Ov8-OVb (SEQ ID NO: 26)TTGAAGCCCTCCACGTGATGGTAC Ov9a (SEQ ID NO: 27) CACACGGTGAACAAGATCACCTTCOv9b (SEQ ID NO: 28) AGTAGCACTGCTCGGAGAAGGTGA Ov10a (SEQ ID NO: 29)ATCTACCACATGGACGAGGAGGAG Ov10b (SEQ ID NO: 30) TGACCAGGTACGGCGTCTCCTCCTOv11a (SEQ ID NO: 31) AGCGCGTCACGCTGGCCGACTTCA Ov11b (SEQ ID NO: 32)TTGCTGAGCACGTTCTTGAAGTCG Ov12a (SEQ ID NO: 33) CACGCCTACAAATTCTTCTTTAAGOv12b (SEQ ID NO: 34) AGTCCTGGTCCATGGACTTAAAGA.The overlapping sequences of the twelve T1R3 overgo probes used in thepresent invention were as follows:

t1r3-OV1a (SEQ ID NO: 35) CTTCCACTCCTGCTGCTACGACTG t1r3-OV1b(SEQ ID NO: 36) TGCCTCGCAGTCCACGCAGTCGTA t1r3-OV2a (SEQ ID NO: 37)AGGTGCGCCGCGTCAAGGGCTTCC t1r3-OV2b (SEQ ID NO: 38)TCGTAGCAGCAGGAGTGGAAGCCC t1r3-OV3a (SEQ ID NO: 39)GTTCCTGGCATGGGGGGAGCCGGC t1r3-OV3b (SEQ ID NO: 40)GAGCAGCACAAGCACAGCCGGCTC t1r3-OV4a (SEQ ID NO: 41)ACAGCCCACTAGTTCAGGCCGCAG t1r3-OV4b (SEQ ID NO: 42)CAGGCCCGGGGTCCCCCTGCGGCC t1r3-OV5a (SEQ ID NO: 43)CCCACTGGTTCAGGCCTCGGGGGG t1r3-OV5b (SEQ ID NO: 44)AAAGCAGGCCAGGGGCCCCCCCGA t1r3-OV6a (SEQ ID NO: 45)AGGCGCTGGTGCACTGCCGCACAC t1r3-OV6b (SEQ ID NO: 46)AAGCTGACCCAGGAGCGTGTGCGG t1r3-OV7a (SEQ ID NO: 47)ACAGAGGCACTGGTGCACTGCCGC t1r3-OV7b (SEQ ID NO: 48)TGATCCAGGAGTGCACGCGGCAGT t1r3-OV8a (SEQID NO: 49)ACCAATGCCACGCTGGCCTTTCTC t1r3-OV8b (SEQ ID NO: 50)AAGTGCCCAGGAAGCAGAGAAAGG t1r3-OV9a (SEQ ID NO: 51)TGGTACATGCTGCCAATGCCACGC t1r3-OV9b (SEQ ID NO: 52)AAGCAGAGGAAAGCCAGCGTGGCA t1r3-0V10a (SEQ ID NO: 53)TACAACCGTGCCCGTGGCCTCACC t1r3-OV10b (SEQ ID NO: 54)AGGCCAGCATGGCGAAGGTGAGGC t1r3-OV11a (SEQ ID NO: 55)TCATCACCTGGGTCTCCTTTGTGC t1r3-OV11b (SEQ ID NO: 56)ACATTGGCCAGGAGGGGCACAAAG t1r3-OV12a (SEQ ID NO: 57)TGCAGATGGGTGCCCTCCTGCTCT t1r3-OV12b (SEQ ID NO: 58)AGGATGCCCAGCACACAGAGCAGG.The single-stranded overhangs were filled in with ³²P labeled dATP anddCTP, and the overgo probes hybridized with BAC libraries.

The overgo strategy is considered to be more versatile than a PCR-basedstrategy by those skilled in the art of comparative physical mapping forthe following reasons: (1) overgo probes are short (e.g., 36mers or40mers), making the probability of good alignment from among manyspecies more favorable; (2) overgo probes are more specific to thetarget genes compared with traditional cDNA and genomic DNA probes usedby PCR; and (3) although overgo probes are short, they are not asrestricted as traditional PCR probes, which cannot tolerate even a fewmismatches, because they can be used in hybridization approaches withBACs or other libraries.

Screening a feline genomic BAC library. Seven DVL1 overgo probes (SEQ IDNOS:21-34) were used in screening a feline genomic BAC library. Probeswere radioactively labeled by the random hexa-nucleotide method(Feinberg & Vogelstein, Analytical Biochemistry, 132:6-13 (1983)).Hybridization and washing of membranes followed standard protocols(Church & Gilbert, PNAS U.S.A., 81:1991-1995 (1984)). Thirty-ninepositive BAC clones were identified. Several BAC ends were sequenced.One clone containing homologous sequence to human chromosome 1p36, BAC552J19, was identified using bioinformatics tools.

Production of a shotgun library for BAC 552J19 and identification of asingle clone containing feline T1R3. BAC DNA from 552J19 was prepared byusing Qiagen Large Construct Kit. DNA was then digested by therestriction enzyme Sau3A1 and subcloned into pGEM+3Z (Promega) vector.After transformants were arrayed to a nylon membrane, two separatehybridizations were performed using seven DVL1 and twelve T1R3 overgoprobes (SEQ ID NOS:35-58). Two clones positive for DVL1 and four clonespositive for T1R3 were found. These clones were confirmed by sequencing.Because DVL1 is the neighboring gene of T1R3 in human and mouse, it islikely this also is the case in cat; therefore, the DVL1 positive clonesverified that the BAC 552J19 is the correct BAC, that is, it is the onecontaining feline T1R3.

Cloning and Characterization of the Feline T1R1 and T1R2 Receptors

Eludication of the cat T1R1 and cat T1R2 receptors also was accomplishedusing an overgo strategy. Overgo probes from conserved coding regionswere designed by aligning T1R1 and T1R2 sequences from many differentspecies, including human, mouse, rat, cow, and pig. The single-strandedoverhangs (14 bases) were filled in with ³²P-labeled dATP and dCTP, andthe overgo probes hybridized with BAC libraries. The overlappingsequences of the six cat T1R1 overgo probes were as follows:

t1r1_1-OVa (SEQ ID NO: 65) TAAACAACTCCACGGCCCTGCTGC t1r1_1-OVb(SEQ ID NO: 66) CCCAGGGTGATGTTGGGCAGCAGG t1r1_2-OVa (SEQ ID NO: 67)GCTGTGTATGCGGTGGCCCATGGC t1r1_2-OVb (SEQ ID NO: 68)CCAGGAGCTGGTGGAGGCCATGGG t1r1_3-OVa (SEQ ID NO: 69)TGCTGACCAACCTGACTGGCAAGG t1r1_3-OVb (SEQ ID NO: 70)TCTGAGGCGACCCACACCTTGCCA t1r1_4-OVa (SEQ ID NO: 71)CCAGTTCAGCTAAACATAAATGAG t1r1_4-OVb (SEQ ID NO: 72)GCCACTGGATTTTGGTCTCATTTA t1r1_5-OVa (SEQ ID NO: 73)AGCTAACACGCTGCTGCTGCTGCT t1r1_5-OVb (SEQ ID NO: 74)AGCAGTCCCAAGCAGCAGCAGCAG t1r1_6-OVa (SEQ ID NO: 75)TGTGTCACCTTCAGCCTGCTCTTC t1r1_6-OVb (SEQ ID NO: 76)TCCAGGACACGAAGTTGAAGAGCA.The overlapping sequences of the seven cat T1R2 overgo probes were asfollows:

t1r2_1-OVa (SEQ ID NO: 77) TACTTCGGCCCCAAGTGCTACATG t1r2_1-OVb(SEQ ID NO: 78) CCGGGTAGAAGAGGATCATGTAGC t1r2_2-OVa (SEQ ID NO: 79)TGGTCACCATCGTGGACCTCTTGG t1r2_2-OVb (SEQ ID NO: 80)AGGTTGAGCACAGTGACCAAGAGG t1r2_3-OVa (SEQ ID NO: 81)ACCAACTACAACGAGGCCAAGTTC t1r2_3-OVb (SEQ ID NO: 82)TCATGCTGAGGGTGATGAACTTGG t1r2_4-OVa (SEQ ID NO: 83)TCCGAGTCCTGGGCCATCGACCCG t1r2_4-OVb (SEQ ID NO: 84)TGAGGTTGTGCAGGACCGGGTCGA t1r2_5-OVa (SEQ ID NO: 85)TACAACCTCATGCAGGCCATGCGC t1r2_5-OVb (SEQ ID NO: 86)TCTCCTCCACCGCGAAGCGCATGG t1r2_6-OVa (SEQ ID NO: 87)ATCACCATCCAGAGCGTGCCCATC t1r2_6-OVb (SEQ ID NO: 88)ACTCACTGAAGCCCGGGATGGGCA t1r2_7-OVa (SEQ ID NO: 89)ACCACCACGTCGAGGCCATGGTGC t1r2_7-OVb (SEQ ID NO: 90)AAGTGCAGCATCAGCTGCACCATG.

Screening a feline genomic BAC library. The T1R1 and T1R2 overgo probeswere used to screen a feline genomic BAC library. Probes wereradioactively labeled by the random hexa-nucleotide method (Feinberg &Vogelstein, Analytical Biochemistry, 132(1):6-13 (1983)). Hybridizationand washing of membranes followed standard protocols (Church & Gilbert,PNAS U.S.A., 81:1991-1995 (1984)). Six positive BAC clones for cat T1R1and eight positive BAC clones for cat T1R2 were identified.

Production of shotgun libraries for BACs containing cat T1R1 and T1R2,and identification of small insert clones containing feline T1R1 andT1R2. Two BACs (150M6 and 233G22) containing cat T1R1 and three BACs(93C1, 240H9 and 400B1) containing cat T1R2 were used to prepare BACDNAs using Qiagen Large Construct Kit. BAC DNAs were digested using theenzyme Sau3AI and the digested BAC DNA fragments were subcloned intopGEM+3Z (Promega) vector. After transformants were arrayed to a nylonmembrane, two separate hybridizations were performed using pooled sixT1R1 and seven T1R2 overgo probes. By sequencing positive clones fromshotgun libraries and by using a chromosome walking strategy, the fullcoding region of the cat T1R1 and exon 3 to exon 6 of cat T1R2 wereobtained.

Elucidation of exon 1 and exon 2 of the cat T1R2 by PCR strategy. Sinceexon 1 and exon 2 of cat T1R2 were not present in the three BACsselected above, PCR was performed using degenerate primers designed fromT1R2 alignments from different species (human, rodents, and dog) and catgenomic DNA as template.

Degenerate primers for cat T1R2 exon1 and exon2: PCR product sizeDex1f1: 5′ TCRGACTTCTACCTGCCTGGRGA 3′ (SEQ ID NO: 91)  85 bp Dex1r1: 5′CTTCACGTTGGCATGGAGGG 3′ (SEQ ID NO: 92) Dex1f2: 5′TACCTCCTGGGTGGCCTCTTC 3′ (SEQ ID NO: 93)  66 bp Dex1r2: 5′TCTTGCACwkGGGCACCTGC 3′ (SEQ ID NO: 94) Dex2f1: 5′AGGTGtTGGGCTACAACCTsAT 3′ (SEQ ID NO: 95) 206 bp Dex2r1: 5′GGGCAkGTAGTGGCTGTAGTC 3′ (SEQ ID NO: 96) Dex2f2: 5′GGCTACAACCTsATGCAGGCCA 3′ (SEQ ID NO: 97) 220 bp Dex2r2: 5′GAGTTGTCAGGGCCAATGACCG 3′. (SEQ ID NO: 98)The PCR products were confirmed by sequencing. The feline BAC librarywas then re-screened using PCR products and four new BACs were retrieved(4O545, 2J533, 4F220 and 24D448). Using a chromosome walking strategy,the complete sequence of exon 1 and exon 2 from these four BAC cloneswere obtained.

Results

More than 3 kb of genomic sequences containing the open reading framefor domestic cat taste receptor, T1R3, were obtained. Approximately 10kb of genomic sequence containing the open reading frame (ORF) for catT1R1 and approximately 38 kb of genomic sequence containing the openreading frame for cat T1R2 were obtained. FIGS. 6A-D show the genomicsequence of cat T1R1 (SEQ ID NO:59) obtained from BAC sequencing. FIGS.7A-E show the genomic sequence of cat T1R2 (SEQ ID NO:62) obtained fromBAC sequencing. The letter “N” denotes gaps between exons or unknownsequences. FIGS. 1A-1I show the multiple sequence alignment of the cDNAsencoding T1R receptors of domestic cat (T1R1, SEQ ID NO:60; T1R2, SEQ IDNO:63; and T1R3, SEQ ID NO:99) with known nucleotide sequences ofreceptors of the T1R family from human (T1R1, SEQ ID NO:8; T1R2, SEQ IDNO:5; T1R3, SEQ ID NO:11), mouse (T1R1, SEQ ID NO:6; T1R2, SEQ ID NO:3;T1R3, SEQ ID NO:9), and rat (T1R1, SEQ ID NO:7; T1R2, SEQ ID NO:4; T1R3,SEQ ID NO:10). An asterisk (*) indicates a conserved nucleotide positionamong the sequences. A heart (♡) indicates the stop codon of felineT1R2.

FIGS. 2A-D show the deduced amino acid sequences of the feline T1R tastereceptors (T1R1, SEQ ID NO:61; T1R2, SEQ ID NO:64; and T1R3, SEQ IDNO:2) aligned with the amino acid sequences of members of the T1Rreceptor family from human (T1R1, SEQ ID NO:17; T1R2, SEQ ID NO:20;T1R3, SEQ ID NO:12), rat (T1R1, SEQ ID NO:16; T1R2, SEQ ID NO:19; T1R3,SEQ ID NO:14), and mouse (T1R1, SEQ ID NO:15; T1R2, SEQ ID NO:18; T1R3,SEQ ID NO:13). An asterisk (*) indicates a conserved nucleotide positionamong the sequences. A colon (:) indicates an observed conserved aminoacid substitution. A period (.) indicates an observed semi-conservedamino acid substitution. The cat T1R1 is very similar to human androdents in terms of gene structure; however, cat T1R2 predicts a shorterprotein of 391 amino acids compared with the human T1R2, which has 839amino acids. This prediction of a short T1R2 is the result of a stopcodon TAA in exon 3. The deduced amino acid sequence for cat T1R3 (SEQID NO:2) contains four additional amino acids at positions 11-14relative to the homologous T1R3 receptors of mouse (SEQ ID NO:13), human(SEQ ID NO:12), and rat (SEQ ID NO:14). The deduced sequence for catreveals a threonine in position 64, a position equivalent to amino acid60 in mouse, and a leucine at position 59, a position equivalent toposition 55 in mouse. In mouse, amino acid substitutions of a threonineat position 60 and an alanine at position 55, both positions locatedwithin the putative extracellular N-terminal domain of the polypeptide,are present in strains of mice demonstrating low preference for thesweet stimulus saccharin (Bachmanov et al., Chem. Senses, 26:925-933(2001)). Leucine is a conservative substitution for alanine.Accordingly, the amino acid sequence differences of cat and mouse T1R3receptor may account for functional differences that lead to differenttaste preferences between the two species.

FIG. 3 illustrates a phylogenetic tree showing the relatedness of thedomestic cat T1R receptor family to the T1R family of receptorsincluding human, rat, and mouse T1R1, T1R2, and T1R3. The T1R receptorsof the rat and mouse are closely related, while the T1R receptors ofhuman and cat diverge from rat and mouse. Interestingly, the sweetstimuli to which the rat and mouse respond are very similar, whereasthose that stimulate the human and those that stimulate the cat differfrom one another and from those for rat and mouse. For example, humansare unique in their ability to taste most high-intensity sweeteners,while cats find many amino acids attractive but are unable to taste mostcarbohydrate and high-intensity sweeteners. The cat T1R2 diverges fromthat of human, mouse, and rat, which is consistent with the fact thatcat does not show preference for the carbohydrate sweeteners.

FIG. 4 illustrates the predicted conformation of cat T1R3 receptor. Thecat T1R3 receptor is a seven-transmembrane domain receptor. Thestructure of the feline T1R3 receptor was generated through use of theprotein modeling program available online through the EuropeanBioinformatics Institute.

FIG. 5A shows the predicted conformation of cat T1R1, indicating thatthe receptor is a 7-transmembrane-type receptor. FIG. 5B illustrates thepredicted conformation of cat T1R2. Since feline T1R2 is a short protein(391 amino acids), a 7 transmembrane domain protein is not predicted.Without seven transmembrane domains, the cat T1R2 receptor may notinteract appropriately with a dimerization partner, such as T1R3, and/orthe plasma membrane, thereby resulting in the cat's inability to tastesweet carbohydrates. The cat T1R2 may have another function.

Table 4 shows the percent homology among the members of the T1R familyin relation to the cat T1R taste receptors. The portion of Table 1 tothe left of the diagonal (in bold type) shows the percent homology basedon the open reading frame of the nucleotide sequences obtained from FIG.1 for the T1R family among human, cat, rat and mouse. The upper portionto the right of the diagonal (in italic type) shows the percent homologyof the T1R members based on the amino acid sequences of FIG. 2. Cat T1R1shows 84% nucleotide sequence homology with human T1R1, 78% with ratT1R1 and 79% with mouse T1R1. At the amino acid level, cat T1R1 shows81% homology with human T1R1, 74% with rat, and 74% with mouse. Cat T1R1shows generally low homology with the other known members of the T1Rfamily, T1R2 and T1R3, from human, rat and mouse. The same range ofrelatively low homology is present among the human, rat and mouse T1R1,T1R2 and T1R3 receptors from the same species. Cat T1R2 shows 72%nucleotide sequence homology with human T1R2, 61% with rat T1R2 and 64%with mouse T1R2. At the amino acid level, cat T1R2 shows 58% homologywith human T1R2, 52% with rat, and 53% with mouse. Since cat T1R2 has ashorter protein (391aa) due to a stop codon in exon 3, cat T1R2 showsmuch lower homology with T1R2 in other species than the homology forT1R1 and T1R3 among different species, which indicates that cat T1R2 isvery different from that of the other species. This is also consistentwith the behavioral responses showing that cats do not show preferencefor carbohydrate sweeteners. This indicates that cat T1R2 may not befunctional, freeing it from selective pressure. Therefore mutations incat T1R2 most likely have accumulated. Cat T1R2 shows generally lowhomology with the other members of the T1R family, T1R1 and T1R3, fromhuman, rat and mouse. The same range of relatively low homology ispresent among the human, rat, and mouse T1R2 and the T1R1 and T1R3receptors from the same species.

TABLE 4 Percent Homology Among Diverse Species for T1Rs Mouse MouseMouse Rat Rat Rat Human Human Human Cat Cat Cat Species T1R1 T1R2 T1R3T1R1 T1R2 T1R3 T1R1 T1R2 T1R3 T1R1 T1R2 T1R3 Mouse 36 30 90 36 30 73 3730 74 30 30 T1R1 Mouse 55 28 36 91 28 34 69 28 36 53 28 T1R2 Mouse 33 1531 28 92 30 27 72 30 25 72 T1R3 Rat 91 55 33 37 31 73 37 31 74 26 31T1R1 Rat 55 91 15 57 28 34 71 29 36 52 28 T1R2 Rat 33 21 93 32 15 31 2773 30 26 72 T1R3 Human 79 56 35 79 56 35 35 31 81 29 31 T1R1 Human 57 7817 56 78 17 57 28 36 58 28 T1R2 Human 41 39 73 39 36 75 40 38 29 23 73T1R3 Cat 79 54 35 78 56 35 84 56 53 28 30 T1R1 Cat 42 64 22 41 61 22 4472 48 44 29 T1R2 Cat 33 34 74 36 36 75 53 39 79 53 39 T1R3 Note: Upperright cells (italics) contain deduced amino acid homology; lower leftcells (bold) contain nucleotide homology.

1. An isolated and purified polynucleotide encoding a T1R receptor, saidpolynucleotide comprising the nucleotide sequence of SEQ ID NO:1 or SEQID NO:99.
 2. The polynucleotide of claim 1, wherein said polynucleotideis DNA.
 3. The polynucleotide of claim 1, wherein said polynucleotide isRNA.
 4. An isolated and purified polynucleotide encoding a T1R receptorpolypeptide comprising an amino acid sequence of SEQ ID NO:2 having anonconserved amino acid substitution at residue 59 or residue
 64. 5. Anexpression vector comprising the polynucleotide of claim 1 operablylinked to a promoter.
 6. A host cell comprising the expression vector ofclaim
 5. 7. The host cell of claim 6 wherein said cell is mammalian. 8.The host cell of claim 6 wherein said cell is a human, murine, or felinecell.
 9. The host cell of claim 6 wherein said cell is bacterial.
 10. Acell culture comprising at least one cell of claim
 6. 11. An isolatedand purified T1R receptor polypeptide comprising: a) an amino acidsequence of SEQ ID NO:2; b) a fragment of at least 30 contiguous aminoacids of SEQ ID NO:2; or c) an amino acid sequence of SEQ ID NO:2 havinga nonconserved amino acid substitution at residue 59 or residue
 64. 12.An isolated and purified T1R receptor comprising the polypeptide ofclaim
 11. 13. The T1R receptor of claim 12 wherein said receptor is ahomodimer.
 14. The T1R receptor of claim 12 wherein said receptor is aheterodimer.
 15. The T1R receptor of claim 12 further comprising apolypeptide comprising the amino acid sequence of SEQ ID NO:61 or asecond polypeptide comprising the amino acid sequence of SEQ ID NO:2.16. A kit for the detection of a polynucleotide encoding a feline T1Rreceptor comprising a polynucleotide that specifically hybridizes to apolynucleotide encoding the polypeptide of claim 11 and instructionsrelating to detection of said polynucleotide.
 17. A method of producinga feline T1R receptor comprising culturing the host cell of claim 6 andrecovering said receptor from said host cell.
 18. The feline T1Rreceptor produced according to the method of claim
 17. 19. A method foridentifying compounds that interact with a feline T1R receptorcomprising: contacting a T1R receptor comprising the polypeptide ofclaim 11 with a test compound, and detecting interaction between saidreceptor and said compound.
 20. The method of claim 19, wherein saidreceptor is bound to a solid support.
 21. The method of claim 20,wherein said solid support is formulated into a feline-specificelectronic tongue.
 22. A method for identifying an agonist of a felineT1R receptor comprising: expressing the polynucleotide of claim 1 in thepresence of a test compound, and detecting an increase in biologicalactivity of a receptor comprising a polypeptide produced by saidexpression step in the presence of said compound relative to biologicalactivity of said receptor in the absence of said compound.
 23. Themethod of claim 22 wherein said receptor is bound to a solid support.24. The method of claim 23 wherein said solid support is formulated intoa feline-specific electronic tongue.
 25. A method for identifying anagonist of a feline T1R receptor comprising: contacting a receptorcomprising the polypeptide of claim 11 with a test compound, anddetecting an increase in biological activity of said receptor in thepresence of said compound relative to biological activity of saidreceptor in the absence of said compound.
 26. The method of claim 25wherein said receptor is bound to a solid support.
 27. The method ofclaim 26 wherein said solid support is formulated into a feline-specificelectronic tongue.
 28. A method for identifying an antagonist of afeline T1R receptor comprising: expressing the polynucleotide of claim 1in the presence of a test compound, and detecting a decrease inbiological activity of a receptor comprising a polypeptide produced bysaid expression step in the presence of said compound relative tobiological activity of said receptor in the absence of said compound.29. The method of claim 28 wherein said receptor is bound to a solidsupport.
 30. The method of claim 29 wherein said solid support isformulated into a feline-specific electronic tongue.
 31. A method foridentifying an antagonist of a feline T1R receptor comprising:contacting a receptor comprising the polypeptide of claim 11 with a testcompound, and detecting a decrease in biological activity of saidreceptor in the presence of said compound relative to biologicalactivity of said receptor in the absence of said compound.
 32. Themethod of claim 31 wherein said receptor is bound to a solid support.33. The method of claim 32 wherein said solid support is formulated intoa feline-specific electronic tongue.
 34. An isolated and purified T1Rreceptor comprising at least one extracellular domain of a feline T1Rreceptor polypeptide, said extracellular domain comprising amino acids1-571 of SEQ ID NO:2, amino acids 628-641 of SEQ ID NO:2, amino acids705-731 of SEQ ID NO:2, or amino acids 787-794 of SEQ ID NO:2.
 35. TheT1R receptor of claim 34, wherein said receptor is a chimeric receptor.36. An isolated and purified T1R receptor comprising at least onetransmembrane domain of a feline T1R receptor polypeptide, saidtransmembrane domain comprising amino acids 572-594 of SEQ ID NO:2,amino acids 610-627 of SEQ ID NO:2, amino acids 642-664 of SEQ ID NO:2,amino acids 681-704 of SEQ ID NO:2, amino acids 731-754 of SEQ ID NO:2,amino acids 767-786 of SEQ ID NO:2, or amino acids 795-812 of SEQ IDNO:2.
 37. The T1R receptor of claim 36, wherein said receptor is achimeric receptor.
 38. An isolated and purified T1R receptor comprisingan intracellular domain of a feline T1R receptor, said intracellulardomain comprising amino acids 595-609 of SEQ ID NO:2, amino acids665-680 of SEQ ID NO:2, amino acids 755-766 of SEQ ID NO:2, or aminoacids 813-865 of SEQ ID NO:2.
 39. The T1R receptor of claim 38, whereinsaid receptor is a chimeric receptor.
 40. An isolated and purifiedantibody that specifically immunoreacts with at least one epitope of thepolypeptide of claim
 11. 41. An isolated and purified antibody thatspecifically immunoreacts with at least one epitope of the T1R receptorof claim 12.